Ransgenic non-human animals for producing heterologous antibodies

ABSTRACT

The invention relates to transgenic non-human animals capable of producing heterologous antibodies and transgenic non-human animals having inactivated endogenous immunoglobulin genes. In one aspect of the invention, endogenous immunoglobulin genes are suppressed by antisense polynucleotides and/or by antiserum directed against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of that species of non-human animal. In one aspect of the invention, one or more transgenes containing sequences of unrearranged heterologous human immunoglobulin heavy chains are introduced into a non-human animal thereby forming a transgenic animal capable of functionally rearranging transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes. Such heterologous human antibodies are produced in B-cells which are thereafter immortalized, e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line capable of producing a monoclonal heterologous antibody. The invention also relates to heavy and light chain immunoglobulin transgenes for making such transgenic non-human animals as well as methods and vectors for disrupting endogenous immunoglobulin loci in the transgenic animal.

This application is a continuation-in-part of U.S. Ser.No. 07/904,068filed 23 Jun. 1992, which is a continuation-in-part of U.S. Ser. No.07/853,408 filed 18 Mar. 1992, which is a continuation-in-part of U.S.Ser. No. 07/810,279 filed Dec. 17, 1991, which is a continuation-in-partof U.S. Ser. No. 07/575,962 filed Aug. 31, 1990 now abandoned, which isa continuation-in-part of U.S. Ser. No. 07/574,748 filed Aug. 29, 1990now abandoned. This application claims foreign priority benefits underTitle 35, United States Code, Section 119, to PCT Application No.PCT/US91/06185 (which corresponds to U.S. Ser. No. 07/834,539 filed Feb.5, 1992).

TECHNICAL FIELD

The invention relates to transgenic non-human animals capable of aproducing heterologous antibodies, transgenes used to produce suchtransgenic animals, transgenes capable of functionally rearranging aheterologous D gene in V-D-J recombination, immortalized B-cells capableof producing heterologous antibodies, methods and transgenes forproducing heterologous antibodies of multiple isotypes, methods andtransgenes for inactivating or suppressing expression of endogenousimmunoglobulin loci, methods and transgenes for producing heterologousantibodies wherein a variable region sequence comprises somatic mutationas compared to germline rearranged variable region sequences, andtransgenic nonhuman animals which produce antibodies having a humanprimary sequence and which bind to human antigens.

BACKGROUND OF THE INVENTION

One of the major impediments facing the development of in vivotherapeutic and diagnostic applications for monoclonal antibodies inhumans is the intrinsic immunogenicity of non-human immunoglobulins. Forexample, when immunocompetent human patients are administeredtherapeutic doses of rodent monoclonal antibodies, the patients produceantibodies against the rodent immunoglobulin sequences; these humananti-mouse antibodies (HAMA) neutralize the therapeutic antibodies andcan cause acute toxicity. Hence, it is desirable to produce humanimmunoglobulins that are reactive with specific human antigens that arepromising therapeutic and/or diagnostic targets. However, producinghuman immunoglobulins that bind specifically with human antigens isproblematic.

The present technology for generating monoclonal antibodies involvespre-exposing, or priming, an animal (usually a rat or mouse) withantigen, harvesting B-cells from that animal, and generating a libraryof hybridoma clones. By screening a hybridoma population for antigenbinding specificity (idiotype) and also screening for immunoglobulinclass (isotype), it is possible to select hybridoma clones that secretethe desired antibody.

However, when present methods for generating monoclonal antibodies areapplied for the purpose of generating human antibodies that have bindingspecificities for human antigens, obtaining B-lymphocytes which producehuman immunoglobulins a serious obstacle, since humans will typicallynot make immune responses against self-antigens.

Hence, present methods of generating human monoclonal antibodies thatare specifically reactive with human antigens are clearly insufficient.It is evident that the same limitations on generating monoclonalantibodies to authentic self antigens apply where non-human species areused as the source of B-cells for making the hybridoma.

The construction of transgenic animals harboring a functionalheterologous immunoglobulin transgene are a method by which antibodiesreactive with self antigens may be produced. However, in order to obtainexpression of therapeutically useful antibodies, or hybridoma clonesproducing such antibodies, the transgenic animal must produce transgenicB cells that are capable of maturing through the B lymphocytedevelopment pathway. Such maturation requires the presence of surfaceIgM on the transgenic B cells, however isotypes other than IgM aredesired for therapeutic uses. Thus, there is a need for transgenes andanimals harboring such transgenes that are able to undergo functionalV-D-J rearrangement to generate recombinational diversity and junctionaldiversity. Further, such transgenes and transgenic animals preferablyinclude cis-acting sequences that facilitate isotype switching from afirst isotype that is required for B cell maturation to a subsequentisotype that has superior therapeutic utility.

A number of experiments have reported the use of transfected cell linesto determine the specific DNA sequences required for Ig generearrangement (reviewed by Lewis and Gellert (1989), Cell, 59,585-588).Such reports have identified putative sequences and concluded that theaccessibility of these sequences to the recombinase enzymes used forrearrangement is modulated by transcription (Yancopoulos and Alt (1985),Cell, 40, 271-281). The sequences for V(D)J joining are reportedly ahighly conserved, near-palindromic heptamer and a less well conservedAT-rich nanomer separated by a spacer of either 12 or 23 bp (Tonegawa(1983), Nature, 302, 575-581; Hesse, et al. (1989), Genes in Dev., 3,1053-1061). Efficient recombination reportedly occurs only between sitescontaining recombination signal sequences with different length spacerregions.

Ig gene rearrangement, though studied in tissue culture cells, has notbeen extensively examined in transgenic mice. Only a handful of reportshave been published describing rearrangement test constructs introducedinto mice [Buchini, et al. (1987), Nature, 326, 409-411 (unrearrangedchicken λ transgene); Goodhart, et al. (1987) , Proc. Natl. Acad. Sci.USA, 84, 4229-4233) (unrearranged rabbit K gene); and Bruggemann, et al.(1989), Proc. Natl. Acad. Sci. USA, 86, 6709-6713 (hybrid mouse-humanheavy chain)]. The results of such experiments, however, have beenvariable, in some cases, producing incomplete or minimal rearrangementof the transgene.

Further, a variety of biological functions of antibody molecules areexerted by the Fc portion of molecules, such as the interaction withmast cells or basophils through Fcε, and binding of complement by Fc μor Fcγ, it further is desirable to generate a functional diversity ofantibodies of a given specificity by variation of isotype.

Although transgenic animals have been generated that incorporatetransgenes encoding one or more chains of a heterologous antibody, therehave been no reports of heterologous transgenes that undergo successfulisotype switching. Transgenic animals that cannot switch isotypes arelimited to producing heterologous antibodies of a single isotype, andmore specifically are limited to producing an isotype that is essentialfor B cell maturation, such as IgM and possibly IgD, which may be oflimited therapeutic utility. Thus, there is a need for heterologousimmunoglobulin transgenes and transgenic animals that are capable ofswitching from an isotype needed for B cell development to an isotypethat has a desired characteristic for therapeutic use.

Based on the foregoing, it is clear that a need exists for methods ofefficiently producing heterologous antibodies, e.g. antibodies encodedby genetic sequences of a first species that are produced in a secondspecies. More particularly, there is a need in the art for heterologousimmunoglobulin transgenes and transgenic animals that are capable ofundergoing functional V-D-J gene rearrangement that incorporates all ora portion of a D gene segment which contributes to recombinationaldiversity. Further, there is a need in the art for transgenes andtransgenic animals that can support V-D-J recombination and isotypeswitching so that (1) functional B cell development may occur, and (2)therapeutically useful heterologous antibodies may be produced. There isalso a need for a source of B cells which can be used to make hybridomasthat produce monoclonal antibodies for therapeutic or diagnostic use inthe particular species for which they are designed. A heterologousimmunoglobulin transgene capable of functional V-D-J recombinationand/or capable of isotype switching could fulfill these needs.

In accordance with the foregoing object transgenic nonhuman animals areprovided which are capable of producing a heterologous antibody, such asa human antibody.

Further, it is an object to provide B-cells from such transgenic animalswhich are capable of expressing heterologous antibodies wherein suchB-cells are immortalized to provide a source of a monoclonal antibodyspecific for a particular antigen.

In accordance with this foregoing object, it is a further object of theinvention to provide hybridoma cells that are capable of producing suchheterologous monoclonal antibodies.

Still further, it is an object herein to provide heterologousunrearranged and rearranged immunoglobulin heavy and light chaintransgenes useful for producing the aforementioned non-human transgenicanimals.

Still further, it is an object herein to provide methods to disruptendogenous immunoglobulin loci in the transgenic animals.

Still further, it is an object herein to provide methods to induceheterologous antibody production in the aforementioned transgenicnon-human animal.

A further object of the invention is to provide methods to generate animmunoglobulin variable region gene segment repertoire that is used toconstruct one or more transgenes of the invention.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

Transgenic nonhuman animals are provided which are capable of producinga heterologous antibody, such as a human antibody. Such heterologousantibodies may be of various isotypes, including: IgG1, IgG2, IgG3,IgG4IgM, IgA1, IgA2, IgAsec, IgD, of IgE. In order for such transgenicnonhuman animals to make an immune response, it is necessary for thetransgenic B cells and pre-B cells to produce surface-boundimmunoglobulin, particularly of the IgM (or possibly IgD) isotype, inorder to effectuate B cell development and antigen-stimulatedmaturation. Such expression of an IgM (or IgD) surface-boundimmunoglobulin is only required during the antigen-stimulated maturationphase of B cell development, and mature B cells may produce otherisotypes, although only a single switched isotype may be produced at atime.

Typically, a cell of the B-cell lineage will produce only a singleisotype at a time, although cis or trans alternative RNA splicing, suchas occurs naturally with the μ_(s) (secreted μ) and μ_(M)(membrane-bound μ) forms, and the μ and δ immunoglobulin chains, maylead to the contemporaneous expression of multiple isotypes by a singlecell. Therefore, in order to produce heterologous antibodies of multipleisotypes, specifically the therapeutically useful IgG, IgA, and IgEisotypes, it is necessary that isotype switching occur. Such isotypeswitching may be classical class-switching or may result from one ormore non-classical isotype switching mechanisms.

The invention provides heterologous immunoglobulin transgenes andtransgenic nonhuman animals harboring such transgenes, wherein thetransgenic animal is capable of producing heterologous antibodies ofmultiple isotypes by undergoing isotype switching. Classical isotypeswitching occurs by recombination events which involve at least oneswitch sequence region in the transgene. Non-classical isotype switchingmay occur by, for example, homologous recombination between humanσ.sub.μ and human Σ.sub.μ sequences (δ-associated deletion). Alternativenon-classical switching mechanisms, such as intertransgene and/orinterchromosomal recombination, among others, may occur and effectuateisotype switching. Such transgenes and transgenic nonhuman animalsproduce a first immunoglobulin isotype that is necessary forantigen-stimulated B cell maturation and can switch to encode andproduce one or more subsequent heterologous isotypes that havetherapeutic and/or diagnostic utility. Transgenic nonhuman animals ofthe invention are thus able to produce, in one embodiment, IgG, IgA,and/or IgE antibodies that are encoded by human immunoglobulin geneticsequences and which also bind specific human antigens with highaffinity.

The invention also encompasses B-cells from such transgenic animals thatare capable of expressing heterologous antibodies of various isotypes,wherein such B-cells are immortalized to provide a source of amonoclonal antibody specific for a particular antigen. Hybridoma cellsthat are derived from such B-cells can serve as one source of suchheterologous monoclonal antibodies.

The invention provides heterologous unrearranged and rearrangedimmunoglobulin heavy and light chain transgenes capable of undergoingisotype switching in vivo in the aforementioned non-human transgenicanimals or in explanted lymphocytes of the B-cell lineage from suchtransgenic animals. Such isotype switching may occur spontaneously or beinduced by treatment of the transgenic animal or explanted Blineagelymphocytes with agents that promote isotype switching, such asT-cell-derived lymphokines (e.g., IL-4 and IFN.sub.γ).

Still further, the invention includes methods to induce heterologousantibody production in the aforementioned transgenic non-human animal,wherein such antibodies may be of various isotypes. These methodsinclude producing an antigenstimulated immune response in a transgenicnonhuman animal for the generation of heterologous antibodies,particularly heterologous antibodies of a switched isotype (i.e., IgG,IgA, and IgE).

This invention provides methods whereby the transgene contains sequencesthat effectuate isotype switching, so that the heterologousimmunoglobulins produced in the transgenic animal and monoclonalantibody clones derived from the B-cells of said animal may be ofvarious isotypes.

This invention further provides methods that facilitate isotypeswitching of the transgene, so that switching between particularisotypes may occur at much higher or lower frequencies or in differenttemporal orders than typically occurs in germline immunoglobulin loci.Switch regions may be grafted from various C_(H) genes and ligated toother C_(H) genes in a transgene construct; such grafted switchsequences will typically function independently of the associated C_(H)gene so that switching in the transgene construct will typically be afunction of the origin of the associated switch regions. Alternatively,or in combination with switch sequences, δ-associated deletion sequencesmay be linked to various C_(H) genes to effect non-classical switchingby deletion of sequences between two δ-associated deletion sequences.Thus, a transgene may be constructed so that a particular C_(H) gene islinked to a different switch sequence and thereby is switched to morefrequently than occurs when the naturally associated switch region isused.

This invention also provides methods to determine whether isotypeswitching of transgene sequences has occurred in a transgenic animalcontaining an immunoglobulin transgene.

The invention provides immunoglobulin transgene constructs and methodsfor producing immunoglobulin transgene constructs, some of which containa subset of germline immunoglobulin loci sequences (which may includedeletions). The invention includes a specific method for facilitatedcloning and construction of immunoglobulin transgenes, involving avector that employs unique XhoI and SalI restriction sites flanked bytwo unique NotI sites. This method exploits the complementary termini ofXhoI and SalI restrictions sites and is useful for creating largeconstructs by ordered concatemerization of restriction fragments in avector.

The transgenes of the invention include a heavy chain transgenecomprising DNA encoding at least one variable gene segment, onediversity gene segment, one joining gene segment and one constant regiongene segment. The immunoglobulin light chain transgene comprises DNAencoding at least one variable gene segment, one joining gene segmentand one constant region gene segment. The gene segments encoding thelight and heavy chain gene segments are heterologous to the transgenicnon-human animal in that they are derived from, or correspond to, DNAencoding immunoglobulin heavy and light chain gene segments from aspecies not consisting of the transgenic non-human animal. In one aspectof the invention, the transgene is constructed such that the individualgene segments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes permit recombination of the gene segments (functionalrearrangement) and expression of the resultant rearranged immunoglobulinheavy and/or light chains within the transgenic non-human animal whensaid animal is exposed to antigen.

In one aspect of the invention, heterologous heavy and lightimmunoglobulin transgenes comprise relatively large fragments ofunrearranged heterologous DNA. Such fragments typically comprise asubstantial portion of the C, J (and in the case of heavy chain, D)segments from a heterologous immunoglobulin locus. In addition, suchfragments also comprise a substantial portion of the variable genesegments.

In one embodiment, such transgene constructs comprise regulatorysequences, e.g. promoters, enhancers, class switch regions,recombination signals and the like, corresponding to sequences derivedfrom the heterologous DNA. Alternatively, such regulatory sequences maybe incorporated into the transgene from the same or a related species ofthe non-human animal used in the invention. For example, humanimmunoglobulin gene segments may be combined in a transgene with arodent immunoglobulin enhancer sequence for use in a transgenic mouse.

In a method of the invention, a transgenic non-human animal containinggermline unrearranged light and heavy immunoglobulin transgenes--thatundergo VDJ joining during D-cell differentiation--is contacted with anantigen to induce production of a heterologous antibody in a secondaryrepertoire B-cell.

Also included in the invention are vectors and methods to disrupt theendogenous immunoglobulin loci in the non-human animal to be used in theinvention. Such vectors and methods utilize a transgene, preferablypositive-negative selection vector, which is constructed such that ittargets the functional disruption of a class of gene segments encoding aheavy and/or light immunoglobulin chain endogenous to the non-humananimal used in the invention. Such endogenous gene segments includediversity, joining and constant region gene segments. In this aspect ofthe invention, the positive-negative selection vector is contacted withat least one embryonic stem cell of a non-human animal after which cellsare selected wherein the positive-negative selection vector hasintegrated into the genome of the non-human animal by way of homologousrecombination. After transplantation, the resultant transgenic non-humananimal is substantially incapable of mounting an immunoglobulin-mediatedimmune response as a result of homologous integration of the vector intochromosomal DNA. Such immune deficient non-human animals may thereafterbe used for study of immune deficiencies or used as the recipient ofheterologous immunoglobulin heavy and light chain transgenes.

The invention also provides vectors, methods, and compositions usefulfor suppressing the expression of one or more species of immunoglobulinchain(s), without disrupting an endogenous immunoglobulin locus. Suchmethods are useful for suppressing expression of one or more endogenousimmunoglobulin chains while permitting the expression of one or moretransgene-encoded immunoglobulin chains. Unlike genetic disruption of anendogenous immunoglobulin chain locus, suppression of immunoglobulinchain expression does not require the time-consuming breeding that isneeded to establish transgenic animals homozygous for a disruptedendogenous Ig locus. An additional advantage of suppression as comparedto engognous Ig gene disruption is that, in certain embodiments, chainsuppression is reversible within an individual animal. For example, Igchain suppression may be accomplished with: (1) transgenes encoding andexpressing antisense RNA that specifically hybridizes to an endogenousIg chain gene sequence, (2) antisense oligonucleotides that specificallyhybridize to an endogenous Ig chain gene sequence, and (3)immunoglobulins that bind specifically to an endogenous Ig chainpolypeptide.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the complementarity determining regions CDR1, CDR2 andCDR3 and framework regions FR1, FR2, FR3 and FR4 in unrearranged genomicDNA and mRNA expressed from a rearranged immunoglobulin heavy chaingene,

FIG. 2 depicts the human λ chain locus,

FIG. 3 depicts the human κ chain locus,

FIG. 4 depicts the human heavy chain locus,

FIG. 5 depicts a transgene construct containing a rearranged IgM geneligated to a 25 kb fragment that contains human γ3 and γ1 constantregions followed by a 700 bp fragment containing the rat chain 3'enhancer sequence.

FIG. 6 is a restriction map of the human κ chain locus depicting thefragments to be used to form a light chain transgene by way of in vivohomologous recombination.

FIG. 7 depicts the construction of pGP1.

FIG. 8 depicts the construction of the polylinker contained in pGP1.

FIG. 9 depicts the fragments used to construct a human heavy chaintransgene of the invention.

FIG. 10 depicts the construction of pHIG1 and pCON1.

FIG. 11 depicts the human Cγ1 fragments which are inserted into pRE3(rat enhancer 3' to form pREG2.

FIG. 12 depicts the construction of pHIG3' and PCON.

FIG. 13 depicts the fragment containing human D region segments used inconstruction of the transgenes of the invention.

FIG. 14 depicts the construction of pHIG2 D segment containing plasmid).

FIG. 15 depicts the fragments covering the human Jκ and human Cκ genesegments used in constructing a transgene of the invention.

FIG. 16 depicts the structure of pE μ.

FIG. 17 depicts the construction of pKapH.

FIGS. 18A through 18D depict the construction of a positive-negativeselection vector for functionally disrupting the endogenous heavy chainimmunoglobulin locus of mouse.

FIGS. 19A through 19C depict the construction of a positive-negativeselection vector for functionally disrupting the endogenousimmunoglobulin light chain loci in mouse.

FIGS. 20 a through e depict the structure of a kappa light chaintargeting vector.

FIGS. 21 a through f depict the structure of a mouse heavy chaintargeting vector.

FIG. 22 depicts the map of vector pGPe.

FIG. 23 depicts the structure of vector pJM2.

FIG. 24 depicts the structure of vector pCOR1.

FIG. 25 depicts the transgene constructs for pIGM1, pHCb1 and pHC2.

FIG. 26 depicts the structure of pγe2.

FIG. 27 depicts the structure of pVGE1.

FIG. 28 depicts the assay results of human Ig expression in a pHC1transgenic mouse.

FIG. 29 depicts the structure of pJCK1.

FIG. 30 depicts the construction of a synthetic heavy chain variableregion.

FIG. 31 is a schematic representation of the heavy chain minilocusconstructs pIGM₁, pHC1, and pHC2.

FIG. 32 is a schematic representation of the heavy chain minilocusconstruct pIGG1 and the κ light chain minilocus construct pKC1, pKVe1,and pKC2.

FIG. 33 depicts a scheme to reconstruct functionally rearranged lightchain genes.

FIG. 34 depicts serum ELISA results

FIG. 35 depicts the results of an ELISA assay of serum from 8 transgenicmice.

FIG. 36 is a schematic representation of plasmid pBCE1.

FIG. 37 (A-C) depicts the immune response of transgenic mice of thepresent invention against KLH-DNP, by measuring IgG and IgM levelsspecific for KLH-DNP (37A), KLH (37B) and BSA-DNP (37C) .

FIG. 38 shows ELISA data demonstrating the presence of antibodies thatbind human carcinoembryonic antigen (CEA) and comprise human μ chains;each panel shows reciprocal serial dilutions from pooled serum samplesobtained from mice on the indicated day following immunization.

FIG. 39 shows ELISA data demonstrating the presence of antibodies thatbind human carcinoembryonic antigen (CEA) and comprise human γ chains;each panel shows reciprocal serial dilutions from pooled serum samplesobtained from mice on the indicated day following immunization.

FIG. 40 shows aligned variable region sequences of 23 randomly-chosencDNAs generated from mRNA obtained from lymphoid tissue of HC1transgenic mice immunized with human carcinoembryonic antigen (CEA) ascompared to the germline transgene sequence (top line); on each linenucleotide changes relative to germline sequence are shown above thealteration in deduced amino acid sequence (if any); the regionscorresponding to heavy chain CDR1, CDR2, and CDR3 are indicated.Non-germline encoded nucleotides are shown in capital letters. GermlineV_(H) 251 and J_(H) are shown in lower case letters. Deduced amino acidchanges are given beneath nucleotide sequences using the conventionalsingle-letter notation.

FIG. 41 show the nucleotide sequence of a human DNA fragment, designatedvk65.3, containing a V.sub.κ gene segment; the deduced amino acidsequences of the V.sub.κ coding regions are also shown; splicing andrecombination signal sequences (heptamer/nonamer) are shown boxed.

FIG. 42 show the nucleotide sequence of a human DNA fragment, designatedvk65.5, containing a V.sub.κ gene segment; the deduced amino acidsequences of the V.sub.κ coding regions are also shown; splicing andrecombination signal sequences (heptamer/nonamer) are shown boxed.

FIG. 43 show the nucleotide sequence of a human DNA fragment, designatedvk65.8, containing a V.sub.κ gene segment; the deduced amino acidsequences of the V.sub.κ coding regions are also shown; splicing andrecombination signal sequences (heptamer/nonamer) are shown boxed.

FIG. 44 show the nucleotide sequence of a human DNA fragment, designatedvk65.15, containing a V.sub.κ gene segment; the deduced amino acidsequences of the V.sub.κ coding regions are also shown; splicing andrecombination signal sequences (heptamer/nonamer) are shown boxed.

FIG. 45 shows formation of a light chain minilocus by homologousrecombination between two overlapping fragments which were co-injected.

Table 1 depicts the sequence of vector pGPe.

Table 2 depicts the sequence of gene V_(H) 49.8.

Table 3 depicts the detection of human IgM and IgG in the serum oftransgenic mice of this invention.

Table 4 depicts sequences of VDJ joints.

Table 5 depicts the distribution of J segments incorporated into pHC1transgene encoded transcripts to J segments found in adult humanperipheral blood lymphocytes (PBL) .

Table 6 depicts the distribution of D segments incorporated into pHC1transgene encoded transcripts to D segments found in adult humanperipheral blood lymphocytes (PBL) .

Table 7 depicts the length of the CDR3 peptides from transcripts within-frame VDJ joints in the pHC1 transgenic mouse and in human PBL.

Table 8 depicts the predicted amino acid sequences of the VDJ regionsfrom 30 clones analyzed from a pHC1 transgenic.

Table 9 shows transgenic mice of line 112 that were used in theindicated experiments; (+) indicates the presence of the respectivetransgene, (++) indicates that the animal is homozygous for the J^(H) Dknockout transgene.

DETAILED DESCRIPTION

As has been discussed supra, it is desirable to produce humanimmunoglobulins that are reactive with specific human antigens that arepromising therapeutic and/or diagnostic targets. However, producinghuman immunoglobulins that bind specifically with human antigens isproblematic.

First, the immunized animal that serves as the source of B cells mustmake an immune response against the presented antigen. In order for ananimal to make an immune response, the antigen presented must be foreignand the animal must not be tolerant to the antigen. Thus, for example,if it is desired to produce a human monoclonal antibody with an idiotypethat binds to a human protein, self-tolerance will prevent an immunizedhuman from making a substantial immune response to the human protein,since the only epitopes of the antigen that may be immunogenic will bethose that result from polymorphism of the protein within the humanpopulation (allogeneic epitopes).

Second, if the animal that serves as the source of B-cells for forming ahybridoma (a human in the illustrative given example) does make animmune response against an authentic self antigen, a severe autoimmunedisease may result in the animal. Where humans would be used as a sourceof B-cells for a hybridoma, such autoimmunization would be consideredunethical by contemporary standards.

One methodology that can be used to obtain human antibodies that arespecifically reactive with human antigens is the production of atransgenic mouse harboring the human immunoglobulin transgene constructsof this invention. Briefly, transgenes containing all or portions of thehuman immunoglobulin heavy and light chain loci, or transgenescontaining synthetic "miniloci" (described infra, and in copendingapplications U.S. Ser. No. 07/574,748 filed Aug. 29, 1990, U.S. Ser. No.07/575,962 filed Aug. 31, 1990, and PCT/US91/06185 filed Aug. 28, 1991)which comprise essential functional elements of the human heavy andlight chain loci, are employed to produce a transgenic nonhuman animal.Such a transgenic nonhuman animal will have the capacity to produceimmunoglobulin chains that are encoded by human immunoglobulin genes,and additionally will be capable of making an immune response againsthuman antigens. Thus, such transgenic animals can serve as a source ofimmune sera reactive with specified human antigens, and B-cells fromsuch transgenic animals can be fused with myeloma cells to producehybridomas that secrete monoclonal antibodies that are encoded by humanimmunoglobulin genes and which are specifically reactive with humanantigens.

The production of transgenic mice containing various forms ofimmunoglobulin genes has been reported previously. Rearranged mouseimmunoglobulin heavy or light chain genes have been used to producetransgenic mice. In addition, functionally rearranged human Ig genesincluding the μ or γ1 constant region have been expressed in transgenicmice. However, experiments in which the transgene comprises unrearranged(V-D-J or V-J not rearranged) immunoglobulin genes have been variable,in some cases, producing incomplete or minimal rearrangement of thetransgene. However, there are no published examples of either rearrangedor unrearranged immunoglobulin transgenes which undergo successfulisotype switching between C_(H) genes within a transgene.

Definitions

As used herein, the term "antibody" refers to a glycoprotein comprisingat least two light polypeptide chains and two heavy polypeptide chains.Each of the heavy and light polypeptide chains contains a variableregion (generally the amino terminal portion of the polypeptide chain)which contains a binding domain which interacts with antigen. Each ofthe heavy and light polypeptide chains also comprises a constant regionof the polypeptide chains (generally the carboxyl terminal portion)which may mediate the binding of the immunoglobulin to host tissues orfactors including various cells of the immune system, some phagocyticcells and the first component (C1q) of the classical complement system.

As used herein, a "heterologous antibody" is defined in relation to thetransgenic non-human organism producing such an antibody. It is definedas an antibody having an amino acid sequence or an encoding DNA sequencecorresponding to that found in an organism not consisting of thetransgenic non-human animal.

As used herein, a "heterohybrid antibody" refers to an antibody having alight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

As used herein, "isotype" refers to the antibody class (e.g., IgM orIgG₁) that is encoded by heavy chain constant region genes.

As used herein, "isotype switching" refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, "nonswitched isotype" refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the C_(H) gene encoding the nonswitched isotype is typically the firstC_(H) gene immediately downstream from the functionally rearranged VDJgene.

As used herein, the term "switch sequence" refers to those DNA sequencesresponsible for switch recombination. A "switch donor" sequence,typically a μ switch region, will be 5' (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The"switch acceptor" region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, "glycosylation pattern" is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the C_(H) genes of the transgene were derived.

As used herein, "specific binding" refers to the property of theantibody: (1) to bind to a predetermined antigen with an affinity of atleast 1×10⁷ M⁻¹, and (2) to preferentially bind to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen.

The term "naturally-occurring", as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term "rearranged" as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term "unrearranged" or "germline configuration" as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

Transgenic Nonhuman Animals Capable of Producing Heterologous Antibodies

The design of a transgenic non-human animal that responds to foreignantigen stimulation with a heterologous antibody repertoire, requiresthat the heterologous immunoglobulin transgenes contained within thetransgenic animal function correctly throughout the pathway of B-celldevelopment. In a preferred embodiment, correct function of aheterologous heavy chain transgene includes isotype switching.Accordingly, the transgenes of the invention are constructed so as toproduce isotype switching and one or more of the following: (1) highlevel and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

As will be apparent from the following disclosure, not all of theforegoing criteria need be met. For example, in those embodimentswherein the endogenous immunoglobulin loci of the transgenic animal arefunctionally disrupted, the transgene need not activate allelicexclusion. Further, in those embodiments wherein the transgene comprisesa functionally rearranged heavy and/or light chain immunoglobulin gene,the second criteria of functional gene rearrangement is unnecessary, atleast for that transgene which is already rearranged. For background onmolecular immunology, see, Fundamental Immunology, 2nd edition (1989),Paul William E., ed. Raven Press, N.Y., which is incorporated herein byreference.

In one aspect of the invention, transgenic non-human animals areprovided that contain rearranged, unrearranged or a combination ofrearranged and unrearranged heterologous immunoglobulin heavy and lightchain transgenes in the germline of the transgenic animal. Each of theheavy chain transgenes comprises at least one C_(H) gene. In addition,the heavy chain transgene may contain functional isotype switchsequences, which are capable of supporting isotype switching of aheterologous transgene encoding multiple C_(H) genes in Bcells of thetransgenic animal. Such switch sequences may be those which occurnaturally in the germline immunoglobulin locus from the species thatserves as the source of the transgene C_(H) genes, or such switchsequences may be derived from those which occur in the species that isto receive the transgene construct (the transgeneic animal). Forexample, a human transgene construct that is used to produce atransgenic mouse may produce a higher frequency of isotype switchingevents if it incorporates switch sequences similar to those that occurnaturally in the mouse heavy chain locus, as presumably the mouse switchsequences are optimized to function with the mouse switch recombinaseenzyme system, whereas the human switch sequences are not. Switchsequences made be isolated and cloned by conventional cloning methods,or may be synthesized de novo from overlapping syntheticoligonucleotides designed on the basis of published sequence informationrelating to immunoglobulin switch region sequences (Mills et al., Nucl.Acids Res. 18:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642(1989), which are incorporated herein by reference).

For each of the foregoing transgenic animals, functionally rearrangedheterologous heavy and light chain immunoglobulin transgenes are foundin a significant fraction of the B-cells of the transgenic animal (atleast 10 percent).

The transgenes of the invention include a heavy chain transgenecomprising DNA encoding at least one variable gene segment, onediversity gene segment, one joining gene segment and at least oneconstant region gene segment. The immunoglobulin light chain transgenecomprises DNA encoding at least one variable gene segment, one joininggene segment and at least one constant region gene segment. The genesegments encoding the light and heavy chain gene segments areheterologous to the transgenic non-human animal in that they are derivedfrom, or correspond to, DNA encoding immunoglobulin heavy and lightchain gene segments from a species not consisting of the transgenicnon-human animal. In one aspect of the invention, the transgene isconstructed such that the individual gene segments are unrearranged,i.e., not rearranged so as to encode a functional immunoglobulin lightor heavy chain. Such unrearranged transgenes support recombination ofthe V, D, and J gene segments (functional rearrangement) and preferablysupport incorporation of all or a portion of a D region gene segment inthe resultant rearranged immunoglobulin heavy chain within thetransgenic non-human animal when exposed to antigen.

In an alternate embodiment, the transgenes comprise an unrearranged"mini-locus". Such transgenes typically comprise a substantial portionof the C, D, and J segments as well as a subset of the V gene segments.In such transgene constructs, the various regulatory sequences, e.g.promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif.

The invention also includes transgenic animals containing germ linecells having a heavy and light transgene wherein one of the saidtransgenes contains rearranged gene segments with the other containingunrearranged gene segments. In the preferred embodiments, the rearrangedtransgene is a light chain immunoglobulin transgene and the unrearrangedtransgene is a heavy chain immunoglobulin transgene.

The Structure and Generation of Antibodies

The basic structure of all immunoglobulins is based upon a unitconsisting of two light polypeptide chains and two heavy polypeptidechains. Each light chain comprises two regions known as the variablelight chain region and the constant light chain region. Similarly, theimmunoglobulin heavy chain comprises two regions designated the variableheavy chain region and the constant heavy chain region.

The constant region for the heavy or light chain is encoded by genomicsequences referred to as heavy or light constant region gene (C_(H))segments. The use of a particular heavy chain gene segment defines theclass of immunoglobulin. For example, in humans, the μ constant regiongene segments define the IgM class of antibody whereas the use of a γ,γ2, γ3 or γ4 constant region gene segment defines the IgG class ofantibodies as well as the IgG subclasses IgG1 through IgG4. Similarly,the use of a α₁ l or α₂ constant region gene segment defines the IgAclass of antibodies as well as the subclasses IgA1 and IgA2. The δ and εconstant region gene segments define the IgD and IgE antibody classes,respectively.

The variable regions of the heavy and light immunoglobulin chainstogether contain the antigen binding domain of the antibody. Because ofthe need for diversity in this region of the antibody to permit bindingto a wide range of antigens, the DNA encoding the initial or primaryrepertoire variable region comprises a number of different DNA segmentsderived from families of specific variable region gene segments. In thecase of the light chain variable region, such families comprise variable(V) gene segments and joining (J) gene segments. Thus, the initialvariable region of the light chain is encoded by one V gene segment andone J gene segment each selected from the family of V and J genesegments contained in the genomic DNA of the organism. In the case ofthe heavy chain variable region, the DNA encoding the initial or primaryrepertoire variable region of the heavy chain comprises one heavy chainV gene segment, one heavy chain diversity (D) gene segment and one Jgene segment, each selected from the appropriate V, D and J families ofimmunoglobulin gene segments in genomic DNA.

In order to increase the diversity of sequences that contribute toforming antibody binding sites, it is preferable that a heavy chaintransgene include cis-acting sequences that support functional V-D-Jrearrangement that can incorporate all or part of a D region genesequence in a rearranged V-D-J gene sequence. Typically, at least about1 percent of expressed transgene-encoded heavy chains (or mRNAs) includerecognizable D region sequences in the V region. Preferably, at leastabout 10 percent of transgene-encoded V regions include recognizable Dregion sequences, more preferably at least about 30 percent, and mostpreferably more than 50 percent include recognizable D region sequences.

A recognizable D region sequence is generally at least about eightconsecutive nucleotides corresponding to a sequence present in a Dregion gene segment of a heavy chain transgene and/or the amino acidsequence encoded by such D region nucleotide sequence. For example, if atransgene includes the D region gene DHQ52, a transgene-encoded mRNAcontaining the sequence 5' -TAACTGGG-3' located in the V region betweena V gene segment sequence and a J gene segment sequence is recognizableas containing a D region sequence, specifically a DHQ52 sequence.Similarly, for example, if a transgene includes the D region gene DHQ52,a transgeneencoded heavy chain polypeptide containing the amino acidsequence -DAF- located in the V region between a V gene segment aminoacid sequence and a J gene segment amino acid sequence is recognizableas containing a D region sequence, specifically a DHQ52 sequence.

However, because of somatic mutation and N-region addition, some Dregion sequences may be recognizable but may not correspond identicallyto a consecutive D region sequence in the transgene. For example, anucleotide sequence 5'-CTAAXTGGGG-3', where X is A, T, or G, and whichis located in a heavy chain V region and flanked by a V region genesequence and a J region gene sequence, can be recognized ascorresponding to the DHQ52 sequence 5'-CTAACTGGG-3'. Similarly, forexample, the polypeptide sequences -DAFDI-, -DYFDY-, or -GAFDI- locatedin a V region and flanked on the amino-terminal side by an amino acidsequence encoded by a transgene V gene sequence and flanked on thecarboxyterminal side by an amino acid sequence encoded by a transgene Jgene sequence is recognizable as a D region sequence.

Therefore, because somatic mutation and N-region addition can producemutations in sequences derived from a transgene D region, the followingdefinition is provided as a guide for determining the presence of arecognizable D region sequence. An amino acid sequence or nucleotidesequence is recognizable as a D region sequence if: (1) the sequence islocated in a V region and is flanked on one side by a V gene sequence(nucleotide sequence or deduced amino acid sequence) and on the otherside by a J gene sequence (nucleotide sequence or deduced amino acidsequence) and (2) the sequence is substantially identical orsubstantially similar to a known D gene sequence (nucleotide sequence orencoded amino acid sequence).

The term "substantial identity" as used herein denotes a characteristicof a polypeptide sequence or nucleic acid sequence, wherein thepolypeptide sequence has at least 50 percent sequence identity comparedto a reference sequence, and the nucleic acid sequence has at least 70percent sequence identity compared to a reference sequence. Thepercentage of sequence identity is calculated excluding small deletionsor additions which total less than 35 percent of the reference sequence.The reference sequence may be a subset of a larger sequence, such as anentire D gene; however, the reference sequence is at least 8 nucleotideslong in the case of polynucleotides, and at least 3 amino residues longin the case of a polypeptide. Typically, the reference sequence is atleast 8 to 12 nucleotides or at least 3 to 4 amino acids, and preferablythe reference sequence is 12 to 15 nucleotides or more, or at least 5amino acids.

The term "substantial similarity" denotes a characteristic of anpolypeptide sequence, wherein the polypeptide sequence has at least 80percent similarity to a reference sequence. The percentage of sequencesimilarity is calculated by scoring identical amino acids or positionalconservative amino acid substitutions as similar. A positionalconservative amino acid substitution is one that can result from asingle nucleotide substitution; a first amino acid is replaced by asecond amino acid where a codon for the first amino acid and a codon forthe second amino acid can differ by a single nucleotide substitution.Thus, for example, the sequence -Lys-Glu-Arg-Val- is substantiallysimilar to the sequence -Asn-Asp-Ser-Val-, since the codon sequence-AAA-GAA-AGA-GUU- can be mutated to -AAC-GAC-AGC-GUU by introducing only3 substitution mutations, single nucleotide substitutions in three ofthe four original codons. The reference sequence may be a subset of alarger sequence, such as an entire D gene; however, the referencesequence is at least 4 amino residues long. Typically, the referencesequence is at least 5 amino acids, and preferably the referencesequence is 6 amino acids or more.

The Primary Repertoire

The process for generating DNA encoding the heavy and light chainimmunoglobulin genes occurs primarily in developing B-cells. Prior tothe joining of various immunoglobulin gene segments, the V, D, J andconstant (C) gene segments are found, for the most part, in clusters ofV, D, J and C gene segments in the precursors of primary repertoireB-cells. Generally, all of the gene segments for a heavy or light chainare located in relatively close proximity on a single chromosome. Suchgenomic DNA prior to recombination of the various immunoglobulin genesegments is referred to herein as "unrearranged" genomic DNA. DuringB-cell differentiation, one of each of the appropriate family members ofthe V, D, J (or only V and J in the case of light chain genes) genesegments are recombined to form functionally rearranged heavy and lightimmunoglobulin genes. Such functional rearrangement is of the variableregion segments to form DNA encoding a functional variable region. Thisgene segment rearrangement process appears to be sequential. First,heavy chain D-to-J joints are made, followed by heavy chain V-to-DJjoints and light chain V-to-J joints. The DNA encoding this initial formof a functional variable region in a light and/or heavy chain isreferred to as "functionally rearranged DNA" or "rearranged DNA". In thecase of the heavy chain, such DNA is referred to as "rearranged heavychain DNA" and in the case of the light chain, such DNA is referred toas "rearranged light chain DNA". Similar language is used to describethe functional rearrangement of the transgenes of the invention.

The recombination of variable region gene segments to form functionalheavy and light chain variable regions is mediated by recombinationsignal sequences (RSS's ) that flank recombinationally competent V, Dand J segments. RSS's necessary and sufficient to direct recombination,comprise a dyad-symmetric heptamer, an AT-rich nonamer and anintervening spacer region of either 12 or 23 base pairs. These signalsare conserved among the different loci and species that carry out D-J(or V-J) recombination and are functionally interchangeable. SeeOettinger, et al. (1990), Science, 248, 1517-1523 and references citedtherein. The heptamer comprises the sequence CACAGTG or its analoguefollowed by a spacer of unconserved sequence and then a nonamer havingthe sequence ACAAAAACC or its analogue. These sequences are found on theJ, or downstream side, of each V and D gene segment. Immediatelypreceding the germline D and J segments are again two recombinationsignal sequences, first the nonamer and then the heptamer againseparated by an unconserved sequence. The heptameric and nonamericsequences following a V_(L), V_(H) or D segment are complementary tothose preceding the J_(L), D or J_(H) segments with which theyrecombine. The spacers between the heptameric and nonameric sequencesare either 12 base pairs long or between 22 and 24 base pairs long.

In addition to the rearrangement of V, D and J segments, furtherdiversity is generated in the primary repertoire of immunoglobulin heavyand light chain by way of variable recombination between the V and Jsegments in the light chain and between the D and J segments of theheavy chain. Such variable recombination is generated by variation inthe exact place at which such segments are joined. Such variation in thelight chain typically occurs within the last codon of the V gene segmentand the first codon of the J segment. Similar imprecision in joiningoccurs on the heavy chain chromosome between the D and J_(H) segmentsand may extend over as many as 10 nucleotides. Furthermore, severalnucleotides may be inserted between the D and JH and between the V_(H)and D gene segments which are not encoded by genomic DNA. The additionof these nucleotides is known as N-region diversity.

After VJ and/or VDJ rearrangement, transcription of the rearrangedvariable region and one or more constant region gene segments locateddownstream from the rearranged variable region produces a primary RNAtranscript which upon appropriate RNA splicing results in an mRNA whichencodes a full length heavy or light immunoglobulin chain. Such heavyand light chains include a leader signal sequence to effect secretionthrough and/or insertion of the immunoglobulin into the transmembraneregion of the B-cell. The DNA encoding this signal sequence is containedwithin the first exon of the V segment used to form the variable regionof the heavy or light immunoglobulin chain. Appropriate regulatorysequences are also present in the mRNA to control translation of themRNA to produce the encoded heavy and light immunoglobulin polypeptideswhich upon proper association with each other form an antibody molecule.

The net effect of such rearrangements in the variable region genesegments and the variable recombination which may occur during suchjoining, is the production of a primary antibody repertoire. Generally,each B-cell which has differentiated to this stage, produces a singleprimary repertoire antibody. During this differentiation process,cellular events occur which suppress the functional rearrangement ofgene segments other than those contained within the functionallyrearranged Ig gene. The process by which diploid B-cells maintain suchmono-specificity is termed allelic exclusion.

The Secondary Repertoire

B-cell clones expressing immunoglobulins from within the set ofsequences comprising the primary repertoire are immediately available torespond to foreign antigens. Because of the limited diversity generatedby simple VJ and VDJ joining, the antibodies produced by the so-calledprimary response are of relatively low affinity. Two different types ofB-cells make up this initial response: precursors of primaryantibody-forming cells and precursors of secondary repertoire B-cells(Linton et al., Cell 59:1049-1059 (1989)). The first type of B-cellmatures into IgM-secreting plasma cells in response to certain antigens.The other B-cells respond to initial exposure to antigen by entering aT-cell dependent maturation pathway.

During the T-cell dependent maturation of antigen stimulated B-cellclones, the structure of the antibody molecule on the cell surfacechanges in two ways: the constant region switches to a non-IgM subtypeand the sequence of the variable region can be modified by multiplesingle amino acid substitutions to produce a higher affinity antibodymolecule.

As previously indicated, each variable region of a heavy or light Igchain contains an antigen binding domain. It has been determined byamino acid and nucleic acid sequencing that somatic mutation during thesecondary response occurs throughout the V region including the threecomplementary determining regions (CDR1, CDR2 and CDR3) also referred toas hypervariable regions 1, 2 and 3 (Kabat et al. Sequences of Proteinsof Immunological Interest (1991) U.S. Department of Health and HumanServices, Washington, DC, incorporated herein by reference. The CDR1 andCDR2 are located within the variable gene segment whereas the CDR3 islargely the result of recombination between V and J gene segments or V,D and J gene segments. Those portions of the variable region which donot consist of CDR1, 2 or 3 are commonly referred to as frameworkregions designated FR1, FR2, FR3 and FR4. See FIG. 1. Duringhypermutation, the rearranged DNA is mutated to give rise to new cloneswith altered Ig molecules. Those clones with higher affinities for theforeign antigen are selectively expanded by helper T-cells, giving riseto affinity maturation of the expressed antibody. Clonal selectiontypically results in expression of clones containing new mutation withinthe CDR1, 2 and/or 3 regions. However, mutations outside these regionsalso occur which influence the specificity and affinity of the antigenbinding domain.

Transgenic Non-Human Animals Capable of Producing Heterologous Antibody

Transgenic non-human animals in one aspect of the invention are producedby introducing at least one of the immunoglobulin transgenes of theinvention (discussed hereinafter) into a zygote or early embryo of anon-human animal. The non-human animals which are used in the inventiongenerally comprise any mammal which is capable of rearrangingimmunoglobulin gene segments to produce a primary antibody response.Such nonhuman transgenic animals may include, for example, transgenicpigs, transgenic rats, transgenic rabbits, transgenic cattle, and othertransgenic animal species, particularly mammalian species, known in theart. A particularly preferred non-human animal is the mouse or othermembers of the rodent family.

However, the invention is not limited to the use of mice. Rather, anynon-human mammal which is capable of mounting a primary and secondaryantibody response may be used. Such animals include non-human primates,such as chimpanzee, bovine, ovine, and porcine species, other members ofthe rodent family, e.g. rat, as well as rabbit and guinea pig.Particular preferred animals are mouse, rat, rabbit and guinea pig, mostpreferably mouse.

In one embodiment of the invention, various gene segments from the humangenome are used in heavy and light chain transgenes in an unrearrangedform. In this embodiment, such transgenes are introduced into mice. Theunrearranged gene segments of the light and/or heavy chain transgenehave DNA sequences unique to the human species which are distinguishablefrom the endogenous immunoglobulin gene segments in the mouse genome.They may be readily detected in unrearranged form in the germ line andsomatic cells not consisting of B-cells and in rearranged form inB-cells.

In an alternate embodiment of the invention, the transgenes compriserearranged heavy and/or light immunoglobulin transgenes. Specificsegments of such transgenes corresponding to functionally rearranged VDJor VJ segments, contain immunoglobulin DNA sequences which are alsoclearly distinguishable from the endogenous immunoglobulin gene segmentsin the mouse.

Such differences in DNA sequence are also reflected in the amino acidsequence encoded by such human immunoglobulin transgenes as compared tothose encoded by mouse B-cells. Thus, human immunoglobulin amino acidsequences may be detected in the transgenic non-human animals of theinvention with antibodies specific for immunoglobulin epitopes encodedby human immunoglobulin gene segments.

Transgenic B-cells containing unrearranged transgenes from human orother species functionally recombine the appropriate gene segments toform functionally rearranged light and heavy chain variable regions. Itwill be readily apparent that the antibody encoded by such rearrangedtransgenes has a DNA and/or amino acid sequence which is heterologous tothat normally encountered in the nonhuman animal used to practice theinvention.

Unrearranged Transgenes

As used herein, an "unrearranged immunoglobulin heavy chain transgene"comprises DNA encoding at least one variable gene segment, one diversitygene segment, one joining gene segment and one constant region genesegment. Each of the gene segments of said heavy chain transgene arederived from, or has a sequence corresponding to, DNA encodingimmunoglobulin heavy chain gene segments from a species not consistingof the non-human animal into which said transgene is introduced.Similarly, as used herein, an "unrearranged immunoglobulin light chaintransgene" comprises DNA encoding at least one variable gene segment,one joining gene segment and at least one constant region gene segmentwherein each gene segment of said light chain transgene is derived from,or has a sequence corresponding to, DNA encoding immunoglobulin lightchain gene segments from a species not consisting of the non-humananimal into which said light chain transgene is introduced.

Such heavy and light chain transgenes in this aspect of the inventioncontain the above-identified gene segments in an unrearranged form.Thus, interposed between the V, D and J segments in the heavy chaintransgene and between the V and J segments on the light chain transgeneare appropriate recombination signal sequences (RSS's). In addition,such transgenes also include appropriate RNA splicing signals to join aconstant region gene segment with the VJ or VDJ rearranged variableregion.

In order to facilitate isotype switching within a heavy chain transgenecontaining more than one C region gene segment, e.g. Cμ and Cγ1 from thehuman genome, as explained below "switch regions" are incorporatedupstream from each of the constant region gene segments and downstreamfrom the variable region gene segments to permit recombination betweensuch constant regions to allow for immunoglobulin class switching, e.g.from IgM to IgG. Such heavy and light immunoglobulin transgenes alsocontain transcription control sequences including promoter regionssituated upstream from the variable region gene segments which typicallycontain TATA motifs. A promoter region can be defined approximately as aDNA sequence that, when operably linked to a downstream sequence, canproduce transcription of the downstream sequence. Promoters may requirethe presence of additional linked cis-acting sequences in order toproduce efficient transcription. In addition, other sequences thatparticipate in the transcription of sterile transcripts are preferablyincluded. Examples of sequences that participate in expression ofsterile transcripts can be found in the published literature, includingRothman et al., Intl. Immunol. 2:621-627 (1990); Reid et al., Proc.Natl. Acad. Sci. USA 86:840-844 (1989); Stavnezer et al., Proc. Natl.Acad. Sci. USA 85:7704-7708 (1988); and Mills et al., Nucl. Acids Res.18:7305-7316 (1991), each of which is incorporated herein by reference.These sequences typically include about at least 50 bp immediatelyupstream of a switch region, preferably about at least 200 bp upstreamof a switch region; and more preferably about at least 200-1000 bp ormore upstream of a switch region. Suitable sequences occur immediatelyupstream of the human S.sub.γ1, S.sub.γ2, S.sub.γ3, S.sub.γ4, S.sub.α1,S.sub.α2, and S.sub.ε switch regions, although the sequences immediatelyupstream of the human S.sub.γ1, and S.sub.γ3 switch regions arepreferable. In particular, interferon (IFN) inducible transcriptionalregulatory elements, such as IFN-inducible enhancers, are preferablyincluded immediately upstream of transgene switch sequences.

In addition to promoters, other regulatory sequences which functionprimarily in B-lineage cells are used. Thus, for example, a light chainenhancer sequence situated preferably between the J and constant regiongene segments on the light chain transgene is used to enhance transgeneexpression, thereby facilitating allelic exclusion. In the case of theheavy chain transgene, regulatory enhancers and also employed. Suchregulatory sequences are used to maximize the transcription andtranslation of the transgene so as to induce allelic exclusion and toprovide relatively high levels of transgene expression.

Although the foregoing promoter and enhancer regulatory controlsequences have been generically described, such regulatory sequences maybe heterologous to the nonhuman animal being derived from the genomicDNA from which the heterologous transgene immunoglobulin gene segmentsare obtained. Alternately, such regulatory gene segments are derivedfrom the corresponding regulatory sequences in the genome of thenon-human animal, or closely related species, which contains the heavyand light transgene.

In the preferred embodiments, gene segments are derived from humanbeings. The transgenic non-human animals harboring such heavy and lighttransgenes are capable of mounting an Ig-mediated immune response to aspecific antigen administered to such an animal. B-cells are producedwithin such an animal which are capable of producing heterologous humanantibody. After immortalization, and the selection for an appropriatemonoclonal antibody (Mab), e.g. a hybridoma, a source of therapeutichuman monoclonal antibody is provided. Such human Mabs havesignificantly reduced immunogenicity when therapeutically administeredto humans.

Although the preferred embodiments disclose the construction of heavyand light transgenes containing human gene segments, the invention isnot so limited. In this regard, it is to be understood that theteachings described herein may be readily adapted to utilizeimmunoglobulin gene segments from a species other than human beings. Forexample, in addition to the therapeutic treatment of humans with theantibodies of the invention, therapeutic antibodies encoded byappropriate gene segments may be utilized to generate monoclonalantibodies for use in the veterinary sciences.

Rearranged Transgenes

In an alternative embodiment, transgenic nonhuman animals containfunctionally at least one rearranged heterologous heavy chainimmunoglobulin transgene in the germline of the transgenic animal. Suchanimals contain primary repertoire B-cells that express such rearrangedheavy transgenes. Such B-cells preferably are capable of undergoingsomatic mutation when contacted with an antigen to form a heterologousantibody having high affinity and specificity for the antigen. Saidrearranged transgenes will contain at least two C_(H) genes and theassociated sequences required for isotype switching.

The invention also includes transgenic animals containing germ linecells having heavy and light transgenes wherein one of the saidtransgenes contains rearranged gene segments with the other containingunrearranged gene segments. In such animals, the heavy chain transgenesshall have at least two C_(H) genes and the associated sequencesrequired for isotype switching.

The invention further includes methods for generating a syntheticvariable region gene segment repertoire to be used in the transgenes ofthe invention. The method comprises generating a population ofimmunoglobulin V segment DNAs wherein each of the V segment DNAs encodesan immunoglobulin V segment and contains at each end a cleavagerecognition site of a restriction endonuclease. The population ofimmunoglobulin V segment DNAs is thereafter concatenated to form thesynthetic immunoglobulin V segment repertoire. Such synthetic variableregion heavy chain transgenes shall have at least two C_(H) genes andthe associated sequences required for isotype switching.

Isotype Switching

In the development of a B lymphocyte, the cell initially produces IgMwith a binding specificity determined by the productively rearrangedV_(H) and V_(L) regions. Subsequently, each B cell and its progeny cellssynthesize antibodies with the same L and H chain V regions, but theymay switch the isotype of the H chain.

The use of μ or δ constant regions is largely determined by alternatesplicing, permitting IgM and IgD to be coexpressed in a single cell. Theother heavy chain isotypes (γ, α, and ε) are only expressed nativelyafter a gene rearrangement event deletes the Cμ and Cδ exons. This generearrangement process, termed isotype switching, typically occurs byrecombination between so called switch segments located immediatelyupstream of each heavy chain gene (except δ). The individual switchsegments are between 2 and 10 kb in length, and consist primarily ofshort repeated sequences. The exact point of recombination differs forindividual class switching events. Investigations which have usedsolution hybridization kinetics or Southern blotting with cDNA-derivedC_(H) probes have confirmed that switching can be associated with lossof C_(H) sequences from the cell.

The switch (S) region of the μ gene, S.sub.μ, is located about 1 to 2 kb5' to the coding sequence and is composed of numerous tandem repeats ofsequences of the form (GAGCT)_(n) (GGGGT), where n is usually 2 to 5 butcan range as high as 17. (See T. Nikaido et al. Nature 292:845-848(1981))

Similar internally repetitive switch sequences spanning severalkilobases have been found 5' of the other C_(H) genes. The Sα region hasbeen sequenced and found to consist of tandemly repeated 80-bp homologyunits, whereas S.sub.γ2a, S.sub.γ2b, and S.sub.γ3 all contain repeated49-bp homology units very similar to each other. (See, P. Szurek et al.,J. Immunol 135:620-626 (1985) and T. Nikaido et al., J. Biol. Chem.257:7322-7329 (1982), which are incorporated herein by reference.) Allthe sequenced S regions include numerous occurrences of the pentamersGAGCT and GGGGT that are the basic repeated elements of the S.sub.μ gene(T. Nikaido et al., J. Biol. Chem. 257:7322-7329 (1982) which isincorporated herein by reference); in the other S regions thesepentamers are not precisely tandemly repeated as in S.sub.μ, but insteadare embedded in larger repeat units. The S.sub.γ1 region has anadditional higher-order structure: two direct repeat sequences flankeach of two clusters of 49-bp tandem repeats. (See M. R. Mowatt et al.,J. Immunol. 136:2674-2683 (1986), which is incorporated herein byreference).

Switch regions of human H chain genes have been found to be very similarto their mouse homologs. Indeed, similarity between pairs of human andmouse clones 5' to the C_(H) genes has been found to be confined to theS regions, a fact that confirms the biological significance of theseregions.

A switch recombination between μ and α genes produces a compositeS.sub.μ -S.sub.α sequence. Typically, there is no specific site, eitherin S.sub.μ or in any other S region, where the recombination alwaysoccurs.

Generally, unlike the enzymatic machinery of V-J recombination, theswitch machinery can apparently accommodate different alignments of therepeated homologous regions of germline S precursors and then join thesequences at different positions within the alignment. (See, T. H.Rabbits et al., Nucleic Acids Res. 9:4509-4524 (1981) and J. Ravetch etal., Proc. Natl. Acad. Sci. USA 77:6734-6738 (1980), which areincorporated herein by reference.)

The exact details of the mechanism(s) of selective activation ofswitching to a particular isotype are unknown. Although exogenousinfluences such as lymphokines and cytokines might upregulateisotype-specific recombinases, it is also possible that the sameenzymatic machinery catalyzes switches to all isotypes and thatspecificity lies in targeting this machinery to specific switch regions.

The T-cell-derived lymphokines IL-4 and IFN.sub.γ have been shown tospecifically promote the expression of certain isotypes: IL-4 decreasesIgM, IgG2a, IgG2b, and IgG3 expression and increases IgE and IgG1expression; while IFN.sub.γ selectively stimulates IgG2a expression andantagonizes the IL-4-induced increase in IgE and IgG1 expression(Coffman et al., J. Immunol. 136:949-954 (1986) and Snapper et al.,Science 236:944-947 (1987), which are incorporated herein by reference).A combination of IL-4 and IL-5 promotes IgA expression (Coffman et al.,J. Immunol. 139:3685-3690 (1987), which is incorporated herein byreference).

Most of the experiments implicating T-cell effects on switching have notruled out the possibility that the observed increase in cells withparticular switch recombinations might reflect selection of preswitchedor precommitted cells; but the most likely explanation is that thelymphokines actually promote switch recombination.

Induction of class switching appears to be associated with steriletranscripts that initiate upstream of the switch segments (Lutzker etal., Mol. Cell. Biol. 8:1849 (1988); Stavnezer et al., Proc. Natl. Acad.Sci. USA 85:7704 (1988); Esser and Radbruch, EMBO J. 8:483 (1989);Berton et al., Proc. Natl. Acad. Sci. USA 86:2829 (1989); Rothman etal., Int. Immunol. 2:621 (1990), each of which is incorporated byreference). For example, the observed induction of the γ1 steriletranscript by IL-4 and inhibition by IFN-γ correlates with theobservation that IL-4 promotes class switching to γ1 in B-cells inculture, while IFN-γ inhibits γ1 expression. Therefore, the inclusion ofregulatory sequences that affect the transcription of steriletranscripts may also affect the rate of isotype switching. For example,increasing the transcription of a particular sterile transcripttypically can be expected to enhance the frequency of isotype switchrecombination involving adjacent switch sequences.

For these reasons, it is preferable that transgenes incorporatetranscriptional regulatory sequences within about 1-2 kb upstream ofeach switch region that is to be utilized for isotype switching. Thesetranscriptional regulatory sequences preferably include a promoter andan enhancer element, and more preferably include the 5' flanking (i.e.,upstream) region that is naturally associated (i.e., occurs in germlineconfiguration) with a switch region. This 5' flanking region istypically about at least 50 nucleotides in length, preferably about atleast 200 nucleotides in length, and more preferably at least 500-1000nucleotides.

Although a 5' flanking sequence from one switch region can be operablylinked to a different switch region for transgene construction (e.g.,the 5' flanking sequence from the human S.sub.γ1 switch can be graftedimmediately upstream of the S.sub.α1 switch), in some embodiments it ispreferred that each switch region incorporated in the transgeneconstruct have the 5' flanking region that occurs immediately upstreamin the naturally occurring germline configuration.

The Transgenic Primary Repertoire

A. The Human Immunoglobulin Loci

An important requirement for transgene function is the generation of aprimary antibody repertoire that is diverse enough to trigger asecondary immune response for a wide range of antigens. The rearrangedheavy chain gene consists of a signal peptide exon, a variable regionexon and a tandem array of multi-domain constant region regions, each ofwhich is encoded by several exons. Each of the constant region genesencode the constant portion of a different class of immunoglobulins.During B-cell development, V region proximal constant regions aredeleted leading to the expression of new heavy chain classes. For eachheavy chain class, alternative patterns of RNA splicing give rise toboth transmembrane and secreted immunoglobulins.

The human heavy chain locus consists of approximately 200 V genesegments spanning 2 Mb, approximately 30 D gene segments spanning about40 kb, six J segments clustered within a 3 kb span, and nine constantregion gene segments spread out over approximately 300 kb. The entirelocus spans approximately 2.5 Mb of the distal portion of the long armof chromosome 14.

B. Gene Fragment Transgenes

1. Heavy Chain Transgene

In a preferred embodiment, immunoglobulin heavy and light chaintransgenes comprise unrearranged genomic DNA from humans. In the case ofthe heavy chain, a preferred transgene comprises a NotI fragment havinga length between 670 to 830 kb. The length of this fragment is ambiguousbecause the 3' restriction site has not been accurately mapped. It isknown, however, to reside between the α1 and ψα gene segments. Thisfragment contains members of all six of the known V_(H) families, the Dand J gene segments, as well as the μ, δ, γ3, γ1 and α1 constant regions(Berman et al., EMBO J. 7:727-738 (1988), which is incorporated hereinby reference). A transgenic mouse line containing this transgenecorrectly expresses a heavy chain class required for B-cell development(IgM) and at least one switched heavy chain class (IgG₁), in conjunctionwith a sufficiently large repertoire of variable regions to trigger asecondary response for most antigens.

2. Light Chain Transgene

A genomic fragment containing all of the necessary gene segments andregulatory sequences from a human light chain locus may be similarlyconstructed. Such transgenes are constructed as described in theExamples and in copending application, entitled "Transgenic Non-HumanAnimals Capable of Producing Heterologous Antibodies," filed August 29,1990, under U.S.S.N. 07/574,748.

C. Transgenes Generated Intracellularly by In vivo Recombination

It is not necessary to isolate the all or part of the heavy chain locuson a single DNA fragment. Thus, for example, the 670-830 kb NotIfragment from the human immunoglobulin heavy chain locus may be formedin vivo in the non-human animal during transgenesis. Such in vivotransgene construction is produced by introducing two or moreoverlapping DNA fragments into an embryonic nucleus of the non-humananimal. The overlapping portions of the DNA fragments have DNA sequenceswhich are substantially homologous. Upon exposure to the recombinasescontained within the embryonic nucleus, the overlapping DNA fragmentshomologously recombined in proper orientation to form the 670-830 kbNotI heavy chain fragment.

In vivo transgene construction can be used to form any number ofimmunoglobulin transgenes which because of their size are otherwisedifficult, or impossible, to make or manipulate by present technology.Thus, in vivo transgene construction is useful to generateimmunoglobulin transgenes which are larger than DNA fragments which maybe manipulated by YAC vectors (Murray and Szostak, Nature 305:189-193(1983)). Such in vivo transgene construction may be used to introduceinto a non-human animal substantially the entire immunoglobulin locifrom a species not consisting of the transgenic non-human animal.

In addition to forming genomic immunoglobulin transgenes, in vivohomologous recombination may also be utilized to form "mini-locus"transgenes as described in the Examples.

In the preferred embodiments utilizing in vivo transgene construction,each overlapping DNA fragment preferably has an overlappingsubstantially homologous DNA sequence between the end portion of one DNAfragment and the end portion of a second DNA fragment. Such overlappingportions of the DNA fragments preferably comprise about 500 bp to about2000 bp, most preferably 1.0 kb to 2.0 kb. Homologous recombination ofoverlapping DNA fragments to form transgenes in vivo is furtherdescribed in commonly assigned U.S. Patent Application entitled"Intracellular Generation of DNA by Homologous Recombination of DNAFragments" filed August 29, 1990, under U.S.S.N. 07/574,747.

D. Minilocus Transgenes

As used herein, the term "immunoglobulin minilocus" refers to a DNAsequence (which may be within a longer sequence), usually of less thanabout 150 kb, typically between about 25 and 100 kb, containing at leastone each of the following: a functional variable (V) gene segment, afunctional joining (J) region segment, at least one functional constant(C) region gene segment, and--if it is a heavy chain minilocus--afunctional diversity (D) region segment, such that said DNA sequencecontains at least one substantial discontinuity (e.g., a deletion,usually of at least about 2 to 5 kb, preferably 10-25 kb or more,relative to the homologous genomic DNA sequence). A light chainminilocus transgene will be at least 25 kb in length, typically 50 to 60kb. A heavy chain transgene will typically be about 70 to 80 kb inlength, preferably at least about 60 kb with two constant regionsoperably linked to switch regions. Furthermore, the individual elementsof the minilocus are preferably in the germline configuration andcapable of undergoing gene rearrangement in the pre-B cell of atransgenic animal so as to express functional antibody molecules withdiverse antigen specificities encoded entirely by the elements of theminilocus. Further, a heavy chain minilocus comprising at least twoC_(H) genes and the requisite switching sequences is typically capableof undergoing isotype switching, so that functional antibody moleculesof different immunoglobulin classes will be generated. Such isotypeswitching may occur in vivo in B-cells residing within the transgenicnonhuman animal, or may occur in cultured cells of the B-cell lineagewhich have been explanted from the transgenic nonhuman animal.

In an alternate preferred embodiment, immunoglobulin heavy chaintransgenes comprise one or more of each of the VH, D, and J_(H) genesegments and two or more of the C_(H) genes. At least one of eachappropriate type gene segment is incorporated into the minilocustransgene. With regard to the C_(H) segments for the heavy chaintransgene, it is preferred that the transgene contain at least one μgene segment and at least one other constant region gene segment, morepreferably a γ gene segment, and most preferably γ3 or γ1. Thispreference is to allow for class switching between IgM and IgG forms ofthe encoded immunoglobulin and the production of a secretable form ofhigh affinity non-IgM immunoglobulin. Other constant region genesegments may also be used such as those which encode for the productionof IgD, IgA and IgE.

Those skilled in the art will also construct transgenes wherein theorder of occurrence of heavy chain C_(H) genes will be different fromthe naturally-occurring spatial order found in the germline of thespecies serving as the donor of the C_(H) genes.

Additionally, those skilled in the art can select C_(H) genes from morethan one individual of a species (e.g., allogeneic C_(H) genes) andincorporate said genes in the transgene as supernumerary C_(H) genescapable of undergoing isotype switching; the resultant transgenicnonhuman animal may then, in some embodiments, make antibodies ofvarious classes including all of the allotypes represented in thespecies from which the transgene C_(H) genes were obtained.

Still further, those skilled in the art can select C_(H) genes fromdifferent species to incorporate into the transgene. Functional switchsequences are included with each C_(H) gene, although the switchsequences used are not necessarily those which occur naturally adjacentto the C_(H) gene. Interspecies C_(H) gene combinations will produce atransgenic nonhuman animal which may produce antibodies of variousclasses corresponding to C_(H) genes from various species. Transgenicnonhuman animals containing interspecies C_(H) transgenes may serve asthe source of B-cells for constructing hybridomas to produce monoclonalsfor veterinary uses.

The heavy chain J region segments in the human comprise six functional Jsegments and three pseudo genes clustered in a 3 kb stretch of DNA.Given its relatively compact size and the ability to isolate thesesegments together with the μ gene and the 5' portion of the δ gene on asingle 23 kb SFiI/SpeI fragment (Sado et al., Biochem. Biophys. Res.Comm. 154:264271 (1988), which is incorporated herein by reference), itis preferred that all of the J region gene segments be used in themini-locus construct. Since this fragment spans the region between the μand δ genes, it is likely to contain all of the 3' cis-linked regulatoryelements required for μ expression. Furthermore, because this fragmentincludes the entire J region, it contains the heavy chain enhancer andthe μ switch region (Mills et al., Nature 306:809 (1983); Yancopoulosand Alt, Ann. Rev. Immunol. 4:339-368 (1986), which are incorporatedherein by reference). It also contains the transcription start siteswhich trigger VDJ joining to form primary repertoire B-cells(Yancopoulos and Alt, Cell 40:271-281 (1985), which is incorporatedherein by reference). Alternatively, a 36 kb BssHII/SpeI1 fragment,which includes part on the D region, may be used in place of the 23 kbSfiI/SpeI1 fragment. The use of such a fragment increases the amount of5' flanking sequence to facilitate efficient D-to-J joining.

The human D region consists of 4 or 5 homologous 9 kb subregions, linkedin tandem (Siebenlist, et al. (1981), Nature, 294, 631-635). Eachsubregion contains up to 10 individual D segments. Some of thesesegments have been mapped and are shown in FIG. 4. Two differentstrategies are used to generate a mini-locus D region. The firststrategy involves using only those D segments located in a shortcontiguous stretch of DNA that includes one or two of the repeated Dsubregions. A candidate is a single 15 kb fragment that contains 12individual D segments. This piece of DNA consists of 2 contiguous EcoRIfragments and has been completely sequenced (Ichihara, et al. (1988),EMBO J., 7, 4141-4150). Twelve D segments should be sufficient for aprimary repertoire. However, given the dispersed nature of the D region,an alternative strategy is to ligate together several non-contiguousD-segment containing fragments, to produce a smaller piece of DNA with agreater number of segments. Additional D-segment genes can beidentified, for example, by the presence of characteristic flankingnonamer and heptamer sequences, supra, and by reference to theliterature.

At least one, and preferably more than one V gene segment is used toconstruct the heavy chain minilocus transgene. Rearranged orunrearranged V segments with or without flanking sequences can beisolated as described in copending applications, U.S.S.N. 07/574,748filed August 29, 1990, PCT/US91/06185 filed August 28, 1991, andU.S.S.N. 7/810,279 filed December 17, 1991, each of which isincorporated herein by reference.

Rearranged or unrearranged V segments, D segments, J segments, and Cgenes, with or without flanking sequences, can be isolated as describedin copending applications U.S.S.N. 7/574,748 filed August 29, 1990 andPCT/US91/06185 filed August 28, 1991.

A minilocus light chain transgene may be similarly constructed from thehuman λ or κ immunoglobulin locus. Thus, for example, an immunoglobulinheavy chain minilocus transgene construct, e.g., of about 75 kb,encoding V, D, J and constant region sequences can be formed from aplurality of DNA fragments, with each sequence being substantiallyhomologous to human gene sequences. Preferably, the sequences areoperably linked to transcription regulatory sequences and are capable ofundergoing rearrangement. With two or more appropriately placed constantregion sequences (e.g., μ and γ) and switch regions, switchrecombination also occurs. An exemplary light chain transgene constructcan be formed similarly from a plurality of DNA fragments, substantiallyhomologous to human DNA and capable of undergoing rearrangement, asdescribed in copending application, U.S.S.N. 7/574,748 filed August 29,1990.

E. Transgene Constructs Capable of Isotype Switching

Ideally, transgene constructs that are intended to undergo classswitching should include all of the cis-acting sequences necessary toregulate sterile transcripts. Naturally occurring switch regions andupstream promoters and regulatory sequences (e.g., IFN-inducibleelements) are preferred cis-acting sequences that are included intransgene constructs capable of isotype switching. About at least 50basepairs, preferably about at least 200 basepairs, and more preferablyat least 500 to 1000 basepairs or more of sequence immediately upstreamof a switch region, preferably a human γ1 switch region, should beoperably linked to a switch sequence, preferably a human γ1 switchsequence. Further, switch regions can be linked upstream of (andadjacent to) C_(H) genes that do not naturally occur next to theparticular switch region. For example, but not for limitation, a humanγ₁ switch region may be linked upstream from a human α₂ C_(H) gene, or amurine γ₁ switch may be linked to a human C_(H) gene. An alternativemethod for obtaining non-classical isotype switching (e.g., δ-associateddeletion) in transgenic mice involves the inclusion of the 400 bp directrepeat sequences (σμ and εμ) that flank the human μ gene (Yasui et al.,Eur. J. Immunol. 19:1399 (1989)). Homologous recombination between thesetwo sequences deletes the μ gene in IgD-only B-cells. Heavy chaintransgenes can be represented by the following formulaic description:

    (V.sub.H).sub.x -(D).sub.y -(J.sub.H).sub.z -(S.sub.D).sub.m -(C.sub.1).sub.n -[(T)-(S.sub.A).sub.p -(C.sub.2)]q

where:

V_(H) is a heavy chain variable region gene segment,

D is a heavy chain D (diversity) region gene segment,

J_(H) is a heavy chain J (joining) region gene segment,

S_(D) is a donor region segment capable of participating in arecombination event with the S_(a) acceptor region segments such thatisotype switching occurs,

C₁ is a heavy chain constant region gene segment encoding an isotypeutilized in for B cell development (e.g., μ or δ),

T is a cis-acting transcriptional regulatory region segment containingat least a promoter,

S_(A) is an acceptor region segment capable of participating in arecombination event with selected S_(D) donor region segments, such thatisotype switching occurs,

C₂ is a heavy chain constant region gene segment encoding an isotypeother than μ (e.g., γ₁, γ₂, γ₃, γ₄, α₁, α₂, ε).

x, y, z, m, n, p, and q are integers. x is 1-100, n is 0-10, y is 1-50,p is 1-10, z is 1-50, q is 0-50, m is 0-10. Typically, when thetransgene is capable of isotype switching, q must be at least 1, m is atleast 1, n is at least 1, and m is greater than or equal to n.

V_(H), D, J_(H), S_(D), C₁, T, S_(A), and C_(z) segments may be selectedfrom various species, preferably mammalian species, and more preferablyfrom human and murine germline DNA.

V_(H) segments may be selected from various species, but are preferablyselected from V_(H) segments that occur naturally in the human germline,such as V_(H251). Typically about 2 V_(H) gene segments are included,preferably about 4 V_(H) segments are included, and most preferably atleast about 10 V_(H) segments are included.

At least one D segment is typically included, although at least 10 Dsegments are preferably included, and some embodiments include more thanten D segments. Some preferred embodiments include human D segments.

Typically at least one J_(H) segment is incorporated in the transgene,although it is preferable to include about six J_(H) segments, and somepreferred embodiments include more than about six J_(H) segments. Somepreferred embodiments include human J_(H) segments, and furtherpreferred embodiments include six human J_(H) segments and no nonhumanJ_(H) segments.

S_(D) segments are donor regions capable of participating inrecombination events with the S_(A) segment of the transgene. Forclassical isotype switching, S_(D) and S_(A) are switch regions such asS.sub.μ, S.sub.γ1, S.sub.γ2, S.sub.γ3, S.sub.γ4, S.sub.α, S.sub.α2, andS.sub.ε. Preferably the switch regions are murine or human, morepreferably S_(D) is a human or murine S.sub.μ and S_(A) is a human ormurine S.sub.γ1. For nonclassical isotype switching (δ-associateddeletion), S_(D) and S_(A) are preferably the 400 basepair direct repeatsequences that flank the human μ gene.

C₁ segments are typically μ or δ genes, preferably a μ gene, and morepreferably a human or murine μ gene.

T segments typically include S' flanking sequences that are adjacent tonaturally occurring (i.e., germline) switch regions. T segmentstypically at least about at least 50 nucleotides in length, preferablyabout at least 200 nucleotides in length, and more preferably at least500-1000 nucleotides in length. Preferably T segments are 5' flankingsequences that occur immediately upstream of human or murine switchregions in a germline configuration. It is also evident to those ofskill in the art that T segments may comprise cis-acting transcriptionalregulatory sequences that do not occur naturally in an animal germline(e.g., viral enhancers and promoters such as those found in SV40,adenovirus, and other viruses that infect eukaryotic cells).

C₂ segments are typically a γ₁, γ₂, γ₃, γ₄, α₁, α₂, or ε C_(H) gene,preferably a human C^(H) gene of these isotypes, and more preferably ahuman γ₁ or γ₃ gene. Murine γ_(2a) and γ_(2b) may also be used, as maydownstream (i.e., switched) isotype genes form various species. Wherethe heavy chain transgene contains an immunoglobulin heavy chainminilocus, the total length of the transgene will be typically 150 kilobasepairs or less.

In general, the transgene will be other than a native heavy chain Iglocus. Thus, for example, deletion of unnecessary regions orsubstitutions with corresponding regions from other species will bepresent.

F. Methods for Determining Functional Isotope Switching in Ig Transgenes

The occurrence of isotype switching in a transgenic nonhuman animal maybe identified by any method known to those in the art. Preferredembodiments include the following, employed either singly or incombination:

1. detection of mRNA transcripts that contain a sequence homologous toat least one transgene downstream C_(H) gene other than δ and anadjacent sequence homologous to a transgene V_(H) -D_(H) -J_(H)rearranged gene; such detection may be by Northern hybridization, S₁nuclease protection assays, PCR amplification, cDNA cloning, or othermethods;

2. detection in the serum of the transgenic animal, or in supernatantsof cultures of hybridoma cells made from B-cells of the transgenicanimal, of immunoglobulin proteins encoded by downstream C_(H) genes,where such proteins can also be shown by immunochemical methods tocomprise a functional variable region;

3. detection, in DNA from B-cells of the transgenic animal or in genomicDNA from hybridoma cells, of DNA rearrangements consistent with theoccurrence of isotype switching in the transgene, such detection may beaccomplished by Southern blot hybridization, PCR amplification, genomiccloning, or other method; or

4. identification of other indicia of isotype switching, such asproduction of sterile transcripts, production of characteristic enzymesinvolved in switching (e.g., "switch recombinase"), or othermanifestations that may be detected, measured, or observed bycontemporary techniques.

Because each transgenic line may represent a different site ofintegration of the transgene, and a potentially different tandem arrayof transgene inserts, and because each different configuration oftransgene and flanking DNA sequences can affect gene expression, it ispreferable to identify and use lines of mice that express high levels ofhuman immunoglobulins, particularly of the IgG isotype, and contain theleast number of copies of the transgene. Single copy transgenicsminimize the potential problem of incomplete allelic expression.Transgenes are typically integrated into host chromosomal DNA, mostusually into germline DNA and propagated by subsequent breeding ofgermline transgenic breeding stock animals. However, other vectors andtransgenic methods known in the present art or subsequently developedmay be substituted as appropriate and as desired by a practitioner.

G. Functional Disruption of Endogenous Immunoglobulin Loci

The expression of successfully rearranged immunoglobulin heavy and lighttransgenes is expected to have a dominant effect by suppressing therearrangement of the endogenous immunoglobulin genes in the transgenicnonhuman animal. However, another way to generate a nonhuman that isdevoid of endogenous antibodies is by mutating the endogenousimmunoglobulin loci. Using embryonic stem cell technology and homologousrecombination, the endogenous immunoglobulin repertoire can be readilyeliminated. The following describes the functional description of themouse immunoglobulin loci. The vectors and methods disclosed, however,can be readily adapted for use in other non-human animals.

Briefly, this technology involves the inactivation of a gene, byhomologous recombination, in a pluripotent cell line that is capable ofdifferentiating into germ cell tissue. A DNA construct that contains analtered, copy of a mouse immunoglobulin gene is introduced into thenuclei of embryonic stem cells. In a portion of the cells, theintroduced DNA recombines with the endogenous copy of the mouse gene,replacing it with the altered copy. Cells containing the newlyengineered genetic lesion are injected into a host mouse embryo, whichis reimplanted into a recipient female. Some of these embryos developinto chimeric mice that possess germ cells entirely derived from themutant cell line. Therefore, by breeding the chimeric mice it ispossible to obtain a new line of mice containing the introduced geneticlesion (reviewed by Capecchi (1989), Science, 244, 1288-1292).

Because the mouse λ locus contributes to only 5% of the immunoglobulins,inactivation of the heavy chain and/or κ-light chain loci is sufficient.There are three ways to disrupt each of these loci, deletion of the Jregion, deletion of the J-C intron enhancer, and disruption of constantregion coding sequences by the introduction of a stop codon. The lastoption is the most straightforward, in terms of DNA construct design.Elimination of the μ gene disrupts B-cell maturation thereby preventingclass switching to any of the functional heavy chain segments. Thestrategy for knocking out these loci is outlined below.

To disrupt the mouse μ and κ genes, targeting vectors are used based onthe design employed by Jaenisch and co-workers (Zijlstra, et al. (1989),Nature, 342, 435-438) for the successful disruption of the mouseβ2-microglobulin gene. The neomycin resistance gene (neo), from theplasmid pMCIneo is inserted into the coding region of the target gene.The pMCIneo insert uses a hybrid viral promoter/enhancer sequence todrive neo expression. This promoter is active in embryonic stem cells.Therefore, neo can be used as a selectable marker for integration of theknock-out construct. The HSV thymidine kinase (tk) gene is added to theend of the construct as a negative selection marker against randominsertion events (Zijlstra, et al., supra.).

A preferred strategy for disrupting the heavy chain locus is theelimination of the J region. This region is fairly compact in the mouse,spanning only 1.3 kb. To construct a gene targeting vector, a 15 kb KpnIfragment containing all of the secreted A constant region exons frommouse genomic library is isolated. The 1.3 kb J region is replaced withthe 1.1 kb insert from pMCIneo. The HSV tk gene is then added to the 5'end of the KpnI fragment. Correct integration of this construct, viahomologous recombination, will result in the replacement of the mouse JHregion with the neogene. Recombinants are screened by PCR, using aprimer based on the neogene and a primer homologous to mouse sequences5' of the KpnI site in the D region.

Alternatively, the heavy-chain locus is knocked out by disrupting thecoding region of the μ gene. This approach involves the same 15 kb KpnIfragment used in the previous approach. The 1.1 kb insert from pMCIneois inserted at a unique BamHI site in exon II, and the HSV tk gene addedto the 3' KpnI end. Double crossover events on either side of the neoinsert, that eliminate the tk gene, are then selected for. These aredetected from pools of selected clones by PCR amplification. One of thePCR primers is derived from neo sequences and the other from mousesequences outside of the targeting vector. The functional disruption ofthe mouse immunoglobulin loci is presented in the Examples.

G. Suppressing Expression of Endogenous Immunoglobulin Loci

In addition to functional disruption of endogenous Ig loci, analternative method for preventing the expression of an endogenous Iglocus is suppression. Suppression of endogenous Ig genes may beaccomplished with antisense RNA produced from one or more integratedtransgenes, by antisense oligonucleotides, and/or by administration ofantisera specific for one or more endogenous Ig chains.

Antisense Polynucleotides

Antisense RNA transgenes can be employed to partially or totallyknock-out expression of specific genes (Pepin et al. (1991) Nature 355:725; Helene., C. and Toulme, J. (1990) Biochimica Biophys. Acta 1049:99; Stout, J. and Caskey, T. (1990) Somat. Cell Mol. Genet. 16: 369;Munir et al. (1990) Somat. Cell Mol. Genet. 16: 383, each of which isincorporated herein by reference).

"Antisense polynucleotides" are polynucleotides that: (1) arecomplementary to all or part of a reference sequence, such as a sequenceof an endogenous Ig C_(H) or C_(L) region, and (2) which specificallyhybridize to a complementary target sequence, such as a chromosomal genelocus or a Ig mRNA. Such complementary antisense polynucleotides mayinclude nucleotide substitutions, additions, deletions, ortranspositions, so long as specific hybridization to the relevant targetsequence is retained as a functional property of the polynucleotide.Complementary antisense polynucleotides include soluble antisense RNA orDNA oligonucleotides which can hybridize specifically to individual mRNAspecies and prevent transcription and/or RNA processing of the mRNAspecies and/or translation of the encoded polypeptide (Ching et al.,Proc. Natl. Acad. Sci. U.S.A. 86:10006-10010 (1989); Broder et al., Ann.Int. Med. 113:604-618 (1990); Loreau et al., FEBS Letters 274:53-56(1990); Holcenberg et al., WO91/11535; U.S.S.N. 07/530,165 ("New humanCRIPTO gene"); WO91/09865; WO91/04753; WO90/13641; and EP 386563, eachof which is incorporated herein by reference). An antisense sequence isa polynucleotide sequence that is complementary to at least oneimmunoglobulin gene sequence of at least about 15 contiguous nucleotidesin length, typically at least 20 to 30 nucleotides in length, andpreferably more than about 30 nucleotides in length. However, in someembodiments, antisense sequences may have substitutions, additions, ordeletions as compared to the complementary immunoglobulin gene sequence,so long as specific hybridization is retained as a property of theantisense polynucleotide. Generally, an antisense sequence iscomplementary to an endogenous immunoglobulin gene sequence thatencodes, or has the potential to encode after DNA rearrangement, animmunoglobulin chain. In some cases, sense sequences corresponding to animmunoglobulin gene sequence may function to suppress expression,particularly by interfering with transcription.

The antisense polynucleotides therefore inhibit production of theencoded polypeptide(s). In this regard, antisense polynucleotides thatinhibit transcription and/or translation of one or more endogenous Igloci can alter the capacity and/or specificity of a non-human animal toproduce immunoglobulin chains encoded by endogenous Ig loci.

Antisense polynucleotides may be produced from a heterologous expressioncassette in a transfectant cell or transgenic cell, such as a transgenicpluripotent hematopoietic stem cell used to reconstitute all or part ofthe hematopoietic stem cell population of an individual, or a transgenicnonhuman animal. Alternatively, the antisense polynucleotides maycomprise soluble oligonucleotides that are administered to the externalmilieu, either in culture medium in vitro or in the circulatory systemor interstitial fluid in vivo. Soluble antisense polynucleotides presentin the external milieu have been shown to gain access to the cytoplasmand inhibit translation of specific mRNA species. In some embodimentsthe antisense polynucleotides comprise methylphosphonate moieties,alternatively phosphorothiolates or O-methylribonucleotides may be used,and chimeric oligonucleotides may also be used (Dagle et al. (1990)Nucleic Acids Res. 18: 4751). For some applications, antisenseoligonucleotides may comprise polyamide nucleic acids (Nielsen et al.(1991) Science 254: 1497). For general methods relating to antisensepolynucleotides, see Antisense RNA and DNA, (1988), D.A. Melton, Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Antisense polynucleotides complementary to one or more sequences areemployed to inhibit transcription, RNA processing, and/or translation ofthe cognate mRNA species and thereby effect a reduction in the amount ofthe respective encoded polypeptide. Such antisense polynucleotides canprovide a therapeutic function by inhibiting the formation of one ormore endogenous Ig chains in vivo.

Whether as soluble antisense oligonucleotides or as antisense RNAtranscribed from an antisense transgene, the antisense polynucleotidesof this invention are selected so as to hybridize preferentially toendogenous Ig sequences at physiological conditions in vivo. Mosttypically, the selected antisense polynucleotides will not appreciablyhybridize to heterologous Ig sequences encoded by a heavy or light chaintransgene of the invention (i.e., the antisense oligonucleotides willnot inhibit transgene Ig expression by more than about 25 to 35percent).

Antiserum Suppression

Partial or complete suppression of endogenous Ig chain expression can beproduced by injecting mice with antisera against one or more endogenousIg chains (Weiss et al. (1984) Proc. Natl. Acad. Sci. (U.S.A.) 81 211,which is incorporated herein by reference). Antisera are selected so asto react specifically with one or more endogenous Ig chains but to haveminimal or no cross-reactivity with heterologous Ig chains encoded by anIg transgene of the invention. Thus, administration of selected antiseraaccording to a schedule as typified by that of Weiss et al. op.cit. willsuppress endogenous Ig chain expression but permits expression ofheterologous Ig chain(s) encoded by a transgene of the presentinvention.

Nucleic Acids

The nucleic acids, the term "substantial homology" indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98 to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form. A nucleic acid is "isolated" or "rendered substantially pure"when purified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures may be mutated, thereof inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where "derived" indicates that asequence is identical or modified from another sequence).

A nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

Specific Preferred Embodiments

A preferred embodiment of the invention is an animal containing at leastone, typically 2-10, and sometimes 25-50 or more copies of the transgenedescribed in Example 12 (e.g., pHC1 or pHC2) bred with an animalcontaining a single copy of a light chain transgene described inExamples 5, 6, 8, or 14, and the offspring bred with the J_(H) deletedanimal described in Example 10. Animals are bred to homozygosity foreach of these three traits. Such animals have the following genotype: asingle copy (per haploid set of chromosomes) of a human heavy chainunrearranged mini-locus (described in Example 12), a single copy (perhaploid set of chromosomes) of a rearranged human κ light chainconstruct (described in Example 14), and a deletion at each endogenousmouse heavy chain locus that removes all of the functional J_(H)segments (described in Example 10). Such animals are bred with mice thatare homozygous for the deletion of the J_(H) segments (Examples 10) toproduce offspring that are homozygous for the J_(H) deletion andhemizygous for the human heavy and light chain constructs. The resultantanimals are injected with antigens and used for production of humanmonoclonal antibodies against these antigens.

B cells isolated from such an animal are monospecific with regard to thehuman heavy and light chains because they contain only a single copy ofeach gene. Furthermore, they will be monospecific with regards to humanor mouse heavy chains because both endogenous mouse heavy chain genecopies are nonfunctional by virtue of the deletion spanning the J_(H)region introduced as described in Example 9 and 12. Furthermore, asubstantial fraction of the B cells will be monospecific with regards tothe human or mouse light chains because expression of the single copy ofthe rearranged human κ light chain gene will allelically andisotypically exclude the rearrangement of the endogenous mouse κ and λchain genes in a significant fraction of B-cells.

The transgenic mouse of the preferred embodiment will exhibitimmunoglobulin production with a significant repertoire, ideallysubstantially similar to that of a native mouse. Thus, for example, inembodiments where the endogenous Ig genes have been inactivated, thetotal immunoglobulin levels will range from about 0.1 to 10 mg/ml ofserum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. Whena transgene capable of effecting a switch to IgG from IgM has beenintroduced into the transgenic mouse, the adult mouse ratio of serum IgGto IgM is preferably about 10:1. 0f course, the IgG to IgM ratio will bemuch lower in the immature mouse In general greater than about 10%,preferably 40 to 80% of the spleen and lymph node B cells expressexclusively human IgG protein.

The repertoire will ideally approximate that shown in a non-transgenicmouse, usually at least about 10% as high, preferably 25 to 50% or more.Generally, at least about a thousand different immunoglobulins (ideallyIgG), preferably 10⁴ to 10⁶ or more, will be produced, dependingprimarily on the number of different V, J and D regions introduced intothe mouse genome. These immunoglobulins will typically recognize aboutone-half or more of highly antigenic proteins, including, but notlimited to: pigeon cytochrome C, chicken lysozyme, pokeweed mitogen,bovine serum albumin, keyhole limpit hemocyanin, influenzahemagglutinin, staphylococcus protein A, sperm whale myoglobin,influenza neuraminidase, and lambda repressor protein. Some of theimmunoglobulins will exhibit an affinity for preselected antigens of atleast about 10⁷ M^(-l), preferably 10⁸ M⁻¹ to 10⁰ M⁻¹ or greater.

Thus, prior to rearrangement of a transgene containing various heavy orlight chain gene segments, such gene segments may be readily identified,e.g. by hybridization or DNA sequencing, as being from a species oforganism other than the transgenic animal.

Although the foregoing describes a preferred embodiment of thetransgenic animal of the invention, other embodiments are defined by thedisclosure herein and more particularly by the transgenes described inthe Examples. Four categories of transgenic animal may be defined:

I. Transgenic animals containing an unrearranged heavy and rearrangedlight immunoglobulin transgene.

II. Transgenic animals containing an unrearranged heavy and unrearrangedlight immunoglobulin transgene

III. Transgenic animal containing rearranged heavy and an unrearrangedlight immunoglobulin transgene, and

IV. Transgenic animals containing rearranged heavy and rearranged lightimmunoglobulin transgenes.

Of these categories of transgenic animal, the preferred order ofpreference is as follows II>I>III>IV where the endogenous light chaingenes (or at least the κ gene) have been knocked out by homologousrecombination (or other method) and I>II>III>IV where the endogenouslight chain genes have not been knocked out and must be dominated byallelic exclusion.

EXPERIMENTAL EXAMPLES METHODS AND MATERIALS

Transgenic mice are derived according to Hogan, et al., "Manipulatingthe Mouse Embryo: A Laboratory Manual", Cold Spring Harbor Laboratory,which is incorporated herein by reference.

Embryonic stem cells are manipulated according to published procedures(Teratocarcinomas and embryonic stem cells: a practical approach, E. J.Robertson, ed., IRL Press, Washington, D.C., 1987; Zjilstra et al.,Nature 342:435-438 (1989); and Schwartzberg et al., Science 246:799-803(1989), each of which is incorporated herein by reference). DNA cloningprocedures are carried out according to J. Sambrook, et al. in MolecularCloning: A Laboratory Manual, 2d ed., 1989, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference.

Oligonucleotides are synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Hybridoma cells and antibodies are manipulated according to "Antibodies:A Laboratory Manual", Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), which is incorporated herein by reference.

EXAMPLE 1 Genomic Heavy Chain Human Ig Transgene

This Example describes the cloning and microinjection of a human genomicheavy chain immunoglobulin transgene which is microinjected into amurine zygote.

Nuclei are isolated from fresh human placental tissue as described byMarzluff et al., "Transcription and Translation: A Practical Approach",B. D. Hammes and S. J. Higgins, eds., pp. 89-129, IRL Press, Oxford(1985)). The isolated nuclei (or PBS washed human spermatocytes) areembedded in a low melting point agarose matrix and lysed with EDTA andproteinase κto expose high molecular weight DNA, which is then digestedin the agarose with the restriction enzyme NotI as described by M.Finney in Current Protocols in Molecular Biology (F. Ausubel, et al.,eds. John Wiley & Sons, Supp. 4, 1988, Section 2.5.1).

The NotI digested DNA is then fractionated by pulsed field gelelectrophoresis as described by Anand et al., Nucl. Acids Res.17:3425-3433 (1989). Fractions enriched for the NotI fragment areassayed by Southern hybridization to detect one or more of the sequencesencoded by this fragment. Such sequences include the heavy chain Dsegments, J segments, μ and γ1 constant regions together withrepresentatives of all 6 VH families (although this fragment isidentified as 670 kb fragment from HeLa cells by Berman et al. (1988),supra., we have found it to be as 830 kb fragment from human placentalan sperm DNA). Those fractions containing this NotI fragment (see FIG.4) are pooled and cloned into the NotI site of the vector pYACNN inYeast cells. Plasmid pYACNN is prepared by digestion of pYAC-4 Neo (Cooket al., Nucleic Acids Res. 16: 11817 (1988)) with EcoRI and ligation inthe presence of the oligonucleotide 5'-AAT TGC GGC CGC-3'.

YAC clones containing the heavy chain NotI fragment are isolated asdescribed by Brownstein et al., Science 244:1348-1351 (1989), and Greenet al., Proc. Natl. Acad. Sci. USA 87:1213-1217 (1990), which areincorporated herein by reference. The cloned NotI insert is isolatedfrom high molecular weight yeast DNA by pulse field gel electrophoresisas described by M. Finney, op cit. The DNA is condensed by the additionof 1 mM spermine and microinjected directly into the nucleus of singlecell embryos previously described.

EXAMPLE 2 Genomic κ Light Chain Human Ig Transgene Formed by In VivoHomologous Recombination

A map of the human κ light chain has been described in Lorenz et al.,Nucl. Acids Res. 15:9667-9677 (1987), which is incorporated herein byreference.

A 450 kb XhoI to NotI fragment that includes all of C.sub.κ, the 3'enhancer, all J segments, and at least five different V segments isisolated and microinjected into the nucleus of single cell embryos asdescribed in Example 1.

EXAMPLE 3 Genomic κ Light Chain Human Ig Transgene Formed by In VivoHomologous Recombination

A 750 kb MluI to NotI fragment that includes all of the above plus atleast 20 more V segments is isolated as described in Example 1 anddigested with BssHII to produce a fragment of about 400 kb.

The 450 kb XhoI to NotI fragment plus the approximately 400 kb MluI toBssHII fragment have sequence overlap defined by the BssHII and XhoIrestriction sites. Homologous recombination of these two fragments uponmicroinjection of a mouse zygote results in a transgene containing atleast an additional 15-20 V segments over that found in the 450 kbXhoI/NotI fragment (Example 2).

EXAMPLE 4 Construction of Heavy Chain Mini-Locus

A. Construction of pGp1 and pGP2

pBR322 is digested with EcoRI and StyI and ligated with the followingoligonucleotides to generate pGP1 which contains a 147 base pair insertcontaining the restriction sites shown in FIG. 8. The generaloverlapping of these oligos is also shown in FIG. 9.

The oligonucleotides are: ##STR1##

This plasmid contains a large polylinker flanked by rare cutting NotIsites for building large inserts that can be isolated from vectorsequences for microinjection. The plasmid is based on pBR322 which isrelatively low copy compared to the pUC based plasmids (pGP1 retains thepBR322 copy number control region near the origin of replication). Lowcopy number reduces the potential toxicity of insert sequences. Inaddition, pGP1 contains a strong transcription terminator sequencederived from trpA (Christie et al., Proc. Natl. Acad. Sci. USA 78:4180(1981)) inserted between the ampicillin resistance gene and thepolylinker. This further reduces the toxicity associated with certaininserts by preventing readthrough transcription coming from theampicillin promoters.

Plasmid pGP2 is derived from pGP1 to introduce an additional restrictionsite (SfiI) in the polylinker. pGP1 is digested with M1uI and SpeI tocut the recognition sequences in the polylinker portion of the plasmid.

The following adapter oligonucleotides are ligated to the thus digestedpGP1 to form pGP2. ##STR2##

pGP2 is identical to pGP1 except that it contains an additional Sfi Isite located between the M1uI and SpeI sites. This allows inserts to becompletely excised with SfiI as well as with NotI.

B. Construction of pRE3 (rat enhancer 3') 70284

An enhancer sequence located downstream of the rat constant region isincluded in the heavy chain constructs.

The heavy chain region 3' enhancer described by Petterson et al., Nature344:165-168 (1990), which is incorporated herein by reference) isisolated and cloned. The rat IGH 3' enhancer sequence is PCR amplifiedby using the following oligonucleotides: ##STR3##

The thus formed double stranded DNA encoding the 3' enhancer is cut withBamHI and SphI and clone into BamHI/SphI cut pGP2 to yield pRE3 (ratenhancer 3').

C. Cloning of Human J-5μμ Region

A substantial portion of this region is cloned by combining two or morefragments isolated from phage lambda inserts. See FIG. 9.

A 6.3 kb BamHI/HindIII fragment that includes all human J segments(Matsuda et al., EMBO J., 7:1047-1051 (1988); Ravetech et al.m Cell,27:583-591 (1981), which are incorporated herein by reference) isisolated from human genomic DNA library using the oligonucleotide GGACTG TGT CCC TGT GTG ATG CTT TTG ATG TCT GGG GCC AAG.

An adjacent 10 kb HindIII/BamII fragment that contains enhancer, switchand constant region coding exons (Yasui et al., Eur. J. Immunol.9:1399-1403 (1989)) is similarly isolated using the oligonucleotide: CACCAA GTT GAC CTG CCT GGT CAC AGA CCT GAC CAC CTA TGA

An adjacent 3' 1.5 kb BamHI fragment is similarly isolated using clonepMUM insert as probe (pMUM is 4 kb EcoRI/HindIII fragment isolated fromhuman genomic DNA library with oligonucleotide: ##STR4## mu membraneexon 1) and cloned into pUC19.

pGP1 is digested with BamHI and Bg1II followed by treatment with calfintestinal alkaline phosphatase.

Fragments (a) and (b) from FIG. 9 are cloned in the digested pGP1. Aclone is then isolated which is oriented such that 5' BamHI site isdestroyed by BamHI/Bg1 fusion. It is identified as pMU (see FIG. 10).pMU is digested with BamHI and fragment (c) from FIG. 9 is inserted. Theorientation is checked with HindIII digest. The resultant plasmid pHIG1(FIG. 10) contains an 18 kb insert encoding J and Cμ segments.

D. Cloning of Cμ Region

pGP1 is digested with BamHI and HindIII is followed by treatment withcalf intestinal alkaline phosphatase (FIG. 14). The so treated fragment(b) of FIG. 14 and fragment (c) of FIG. 14 are cloned into theBamHI/HindIII cut pGP1. Proper orientation of fragment (c) is checked byHindIII digestion to form pCON1 containing a 12 kb insert encoding theCμ region.

Whereas pHIG1 contains J segments, switch and μ sequences in its 18 kbinsert with an SfiI 3' site and a SpeI 5' site in a polylinker flankedby NotI sites, will be used for rearranged VDJ segments. pCON1 isidentical except that it lacks the J region and contains only a 12 kbinsert. The use of pCON1 in the construction of fragment containingrearranged VDJ segments will be described hereinafter.

E. Cloning of γ-1 Constant Region (pREG2)

The cloning of the human γ-1 region is depicted in FIG. 16.

Yamamura et al., Proc. Natl. Acad. Sci. USA 83:2152-2156 (1986) reportedthe expression of membrane bound human γ-1 from a transgene constructthat had been partially deleted on integration. Their results indicatethat the 3' BamHI site delineates a sequence that includes thetransmembrane rearranged and switched copy of the gamma gene with a V-Cintron of less than 5 kb. Therefore, in the unrearranged, unswitchedgene, the entire switch region is included in a sequence beginning lessthan 5 kb from the 5' end of the first γ-1 constant exon. Therefore itis included in the 5' 5.3 kb HindIII fragment (Ellison et al., NucleicAcids Res. 10:4071-4079 (1982), which is incorporated herein byreference). Takahashi et al., Cell 29:671-679 (1982), which isincorporated herein by reference, also reports that this fragmentcontains the switch sequence, and this fragment together with the 7.7 kbHindIII to BamHI fragment must include all of the sequences we need forthe transgene construct. An intronic sequence is a nucleotide sequenceof at least 15 contiguous nucleotides that occurs in an intron of aspecified gene.

Phage clones containing the γ-1 region are identified and isolated usingthe following oligonucleotide which is specific for the third exon ofγ-I (CH3). ##STR5##

A 7.7 kb HindIII to BglII fragment (fragment (a) in FIG. 11) is clonedinto HindIII/BglII cut pRE3 to form pREG1. The upstream 5.3 kb HindIIIfragment (fragment (b) in FIG. 11) is cloned into HindIII digested pREG1to form pREG2. Correct orientation is confirmed by BamHI/SpeI digestion.

F. Combining Cγ and Cμ

The previously described plasmid pHIG1 contains human J segments and theCμ constant region exons. To provide a transgene containing the Cμconstant region gene segments, pHIG1 was digested with SfiI (FIG. 10).The plasmid pREG2 was also digested with SfiI to produce a 13.5 kbinsert containing human Cγ exons and the rat 3' enhancer sequence. Thesesequences were combined to produce the plasmid pHIG3' (FIG. 12)containing the human J segments, the human Cμ constant region, the humanCγ1 constant region and the rat 3' enhancer contained on a 31.5 kbinsert.

A second plasmid encoding human Cμ and human Cγ1 without J segments isconstructed by digesting pCON1 with SfiI and combining that with theSfiI fragment containing the human Cγ region and the rat 3' enhancer bydigesting pREG2 with SfiI. The resultant plasmid, pCON (FIG. 12)contains a 26 kb NotI/SpeI insert containing human Cμ, human γ1 and therat 3' enhancer sequence.

G. Cloning of D Segment

The strategy for cloning the human D segments is depicted in FIG. 13.Phage clones from the human genomic library containing D segments areidentified and isolated using probes specific for diversity regionsequences (Ichihara et al., EMBO J. 7:4141-4150 (1988)). The followingoligonucleotides are used: ##STR6##

A 5.2 kb XhoI fragment (fragment (b) in FIG. 13) containing DLR1, DXP1,DXP'I, and DA1 is isolated from a phage clone identified with oligoDXP1.

A 3.2 kb XbaI fragment (fragment (c) in FIG. 13) containing DXP4, DA4and DK4 is isolated from a phage clone identified with oligo DXP4.

Fragments (b), (c) and (d) from FIG. 13 are combined and cloned into theXbaI/XhoI site of pGP1 to form pHIG2 which contains a 10.6 kb insert.

This cloning is performed sequentially. First, the 5.2 kb fragment (b)in FIG. 13 and the 2.2 kb fragment (d) of FIG. 13 are treated with calfintestinal alkaline phosphatase and cloned into pGP1 digested with XhoIand XbaI. The resultant clones are screened with the 5.2 and 2.2 kbinsert. Half of those clones testing positive with the 5.2 and 2.2 kbinserts have the 5.2 kb insert in the proper orientation as determinedby BamHI digestion. The 3.2 kb XbaI fragment from FIG. 13 is then clonedinto this intermediate plasmid containing fragments (b) and (d) to formpHIG2. This plasmid contains diversity segments cloned into thepolylinker with a unique 5' SfiI site and unique 3' SpeI site. Theentire polylinker is flanked by NotI sites.

H. Construction of Heavy Chain Minilocus

The following describes the construction of a human heavy chainmini-locus which contain one or more V segments.

An unrearranged V segment corresponding to that identified as the Vsegment contained in the hybridoma of Newkirk et al., J. Clin. Invest.81:1511-1518 (1988), which is incorporated herein by reference, isisolated using the following oligonucleotide: ##STR7##

A restriction map of the unrearranged V segment is determined toidentify unique restriction sites which provide upon digestion a DNAfragment having a length approximately 2 kb containing the unrearrangedV segment together with 5' and 3' flanking sequences. The 5' primesequences will include promoter and other regulatory sequences whereasthe 3' flanking sequence provides recombination sequences necessary forV-DJ joining. This approximately 3.0 kb V segment insert is cloned intothe polylinker of pGB2 to form pVH1.

pVH1 is digested with SfiI and the resultant fragment is cloned into theSfiI site of pHIG2 to form a pHIG5'. Since pHIG2 contains D segmentsonly, the resultant pHIG5' plasmid contains a single V segment togetherwith D segments. The size of the insert contained in pHIG5 is 10.6 kbplus the size of the V segment insert.

The insert from pHIG5 is excised by digestion with NotI and SpeI andisolated. pHIG3' which contains J, Cμ and cγ1 segments is digested withSpeI and NotI and the 3' kb fragment containing such sequences and therat 3' enhancer sequence is isolated. These two fragments are combinedand ligated into NotI digested pGP1 to produce pHIG which containsinsert encoding a V segment, nine D segments, six functional J segments,Cμ, Cγ and the rat 3' enhancer. The size of this insert is approximately43 kb plus the size of the V segment insert.

I. Construction of Heavy Chain Minilocus by Homologous Recombination

As indicated in the previous section, the insert of pHIG isapproximately 43 to 45 kb when a single V segment is employed. Thisinsert size is at or near the limit of that which may be readily clonedinto plasmid vectors. In order to provide for the use of a greaternumber of V segments, the following describes in vivo homologousrecombination of overlapping DNA fragments which upon homologousrecombination within a zygote or ES cell form a transgene containing therat 3' enhancer sequence, the human Cμ, the human Cγ1, human J segments,human D segments and a multiplicity of human V segments.

A 6.3 kb BamHI/HindIII fragment containing human J segments (seefragment (a) in FIG. 9) is cloned into MluI/SpeI digested pHIG5' usingthe following adapters: ##STR8##

The resultant is plasmid designated pHIG5'O (overlap). The insertcontained in this plasmid contains human V, D and J segments. When thesingle V segment from pVH1 is used, the size of this insert isapproximately 17 kb plus 2 kb. This insert is isolated and combined withthe insert from pHIG3' which contains the human J, Cμ, γ1 and rat 3'enhancer sequences. Both inserts contain human J segments which providefor approximately 6.3 kb of overlap between the two DNA fragments. Whencoinjected into the mouse zygote, in vivo homologous recombinationoccurs generating a transgene equivalent to the insert contained inpHIG.

This approach provides for the addition of a multiplicity of V segmentsinto the transgene formed in vivo. For example, instead of incorporatinga single V segment into pHIG5', a multiplicity of V segments containedon (1) isolated genomic DNA, (2) ligated DNA derived from genomic DNA,or (3) DNA encoding a synthetic V segment repertoire is cloned intopHIG2 at the SfiI site to generate pHIG5' V_(N). The J segments fragment(a) of FIG. 9 is then cloned into pHIG5' V_(N) and the insert isolated.This insert now contains a multiplicity of V segments and J segmentswhich overlap with the J segments contained on the insert isolated frompHIG3'. When cointroduced into the nucleus of a mouse zygote, homologousrecombination occurs to generate in vivo the transgene encoding multipleV segments and multiple J segments, multiple D segments, the Cμ region,the Cβ1 region (all from human) and the rat 3' enhancer sequence.

EXAMPLE 5 Construction of Light Chain Minilocus

A. Construction of pEμ1

The construction of pEμ1 is depicted in FIG. 16. The mouse heavy chainenhancer is isolated on the XbaI to EcoRI 678 bp fragment (Banerji etal., Cell 33:729-740 (1983)) from phage clones using oligo: ##STR9##

This Eμ fragment is cloned into EcoRV/XbaI digested pGP1 by blunt endfilling in EcoRI site. The resultant plasmid is designated pEmu1.

B. Construction Of κ Light chain Minilocus

The κ construct contains at least one human V.sub.κ segment, all fivehuman J_(K) segments, the human J-C.sub.κ enhancer, human K constantregion exon, and, ideally, the human 3' κ enhancer (Meyer et al., EMBOJ. 8:1959:1959-1964 (1989)). The K enhancer in mouse is 9 kb downstreamfrom C_(K). However, it is as yet unidentified in the human. Inaddition, the construct contains a copy of the mouse heavy chain J-Cμenhancers.

The minilocus is constructed from four component fragments:

(a) A 16 kb SmaI fragment that contains the human C_(K) exon and the 3'human enhancer by analogy with the mouse locus;

(b) A 5' adjacent 5 kb SmaI fragment, which contains all five Jsegments;

(c) The mouse heavy chain intronic enhancer isolated from pEμ1 (thissequence is included to induce expression of the light chain constructas early as possible in B-cell development. Because the heavy chaingenes are transcribed earlier than the light chain genes, this heavychain enhancer is presumably active at an earlier stage than theintronic κ enhancer); and

(d) A fragment containing one or more V segments.

The preparation of this construct is as follows. Human placental DNA isdigested with SmaI and fractionated on agarose gel by electrophoresis.Similarly, human placental DNA is digested with BamHI and fractionatedby electrophoresis. The 16 kb fraction is isolated from the SmaIdigested gel and the 11 kb region is similarly isolated from the gelcontaining DNA digested with BamHI.

The 16 kb SmaI fraction is cloned into Lambda FIX II (Stratagene, LaJolla, Calif.) which has been digested with XhoI, treated with klenowfragment DNA polymerase to fill in the XhoI restriction digest product.Ligation of the 16 kb SmaI fraction destroys the SmaI sites and lasesXhoI sites intact.

The 11 kb BamHI fraction is cloned into γ EMBL3 (Strategene, La Jolla,Calif.) which is digested with BamHI prior to cloning.

Clones from each library were probed with the C_(K) specific oligo:##STR10##

A 16 kb XhoI insert that was subcloned into the XhoI cut pEμ1 so thatC.sub.κ is adjacent to the SmaI site. The resultant plasmid wasdesignated pKapl.

The above C.sub.κ specific oligonucleotide is used to probe the γEMBL3/BamHI library to identify an 11 kb clone. A 5 kb SmaI fragment(fragment (b) in FIG. 20) is subcloned and subsequently inserted intopKap1 digested with SmaI. Those plasmids containing the correctorientation of J segments, C.sub.κ and the Eμ enhancer are designatedpKap2.

One or more Vκ segments are thereafter subcloned into the MluI site ofpKap2 to yield the plasmid pKapH which encodes the human Vκ segments,the human JK segments, the human Cκ segments and the human Eμ enhancer.This insert is excised by digesting pKapH with NotI and purified byagarose gel electrophoresis. The thus purified insert is microinjectedinto the pronucleus of a mouse zygote as previously described.

C. Construction of κ Light Chain Minilocus by In Vivo HomologousRecombination

The 11 kb BamHI fragment is cloned into BamHI digested pGP1 such thatthe 3' end is toward the SfiI site. The resultant plasmid is designatedpKAPint. One or more VK segments is inserted into the polylinker betweenthe BamHI and SpeI sites in pKAPint to form pKapHV. The insert of pKapHVis excised by digestion with NotI and purified. The insert from pKap2 isexcised by digestion with NotI and purified. Each of these fragmentscontain regions of homology in that the fragment from pKapHV contains a5 kb sequence of DNA that include the J.sub.κ segments which issubstantially homologous to the 5 kb SmaI fragment contained in theinsert obtained from pKap2. As such, these inserts are capable ofhomologously recombining when microinjected into a mouse zygote to forma transgene encoding V.sub.κ, J.sub.κ and C.sub.κ.

EXAMPLE 6 Isolation of Genomic Clones Corresponding to Rearranged andExpressed Copies of Immunoglobulin κ Light Chain Genes

This example describes the cloning of immunoglobulin κ light chain genesfrom cultured cells that express an immunoglobulin of interest. Suchcells may contain multiple alleles of a given immunoglobulin gene. Forexample, a hybridoma might contain four copies of the κ light chaingene, two copies from the fusion partner cell line and two copies fromthe original B-cell expressing the immunoglobulin of interest. Of thesefour copies, only one encodes the immunoglobulin of interest, despitethe fact that several of them may be rearranged. The procedure describedin this example allows for the selective cloning of the expressed copyof the κ light chain.

A. Double Stranded cDNA

Cells from human hybridoma, or lymphoma, or other cell line thatsynthesizes either cell surface or secreted or both forms of IgM with aκ light chain are used for the isolation of polyA+RNA. The RNA is thenused for the synthesis of oligo dT primed cDNA using the enzyme reversetranscriptase. The single stranded cDNA is then isolated and G residuesare added to the 3' end using the enzyme polynucleotide terminaltransferase. The Gtailed single-stranded cDNA is then purified and usedas template for second strand synthesis (catalyzed by the enzyme DNApolymerase) using the following oligonucleotide as a primer: ##STR11##

The double stranded cDNA is isolated and used for determining thenucleotide sequence of the 5' end of the mRNAs encoding the heavy andlight chains of the expressed immunoglobulin molecule. Genomic clones ofthese expressed genes are then isolated. The procedure for cloning theexpressed light chain gene is outlined in part B below.

B. Light Chain

The double stranded cDNA described in part A is denatured and used as atemplate for a third round of DNA synthesis using the followingoligonucleotide primer: ##STR12##

This primer contains sequences specific for the constant portion of theκ light chain message (TCA TCA GAT GGC GGG AAG ATG AAG ACA GAT GGT GCA)as well as unique sequences that can be used as a primer for the PCRamplification of the newly synthesized DNA strand (GTA CGC CAT ATC AGCTGG ATG AAG). The sequence is amplified by PCR using the following twooligonucleotide primers: ##STR13##

The PCR amplified sequence is then purified by gel electrophoresis andused as template for dideoxy sequencing reactions using the followingoligonucleotide as a primer: ##STR14##

The first 42 nucleotides of sequence will then be used to synthesize aunique probe for isolating the gene from which immunoglobulin messagewas transcribed. This synthetic nucleotide segment of DNA will bereferred to below as o-kappa.

A Southern blot of DNA, isolated from the Ig expressing cell line anddigested individually and in pairwise combinations with severaldifferent restriction endonucleases including SmaI, is then probed withthe 32-P labelled unique oligonucleotide o-kappa. A unique restrictionendonuclease site is identified upstream of the rearranged V segment.

DNA from the Ig expressing cell line is then cut with SmaI and secondenzyme (or BamHI or KpnI if there is SmaI site inside V segment). Anyresulting non-blunted ends are treated with the enzyme T4 DNA polymeraseto give blunt ended DNA molecules. Then add restriction site encodinglinkers (BamHI, EcoRI or XhoI depending on what site does not exist infragment) and cut with the corresponding linker enzyme to give DNAfragments with BamHI, EcoRI or XhoI ends. The DNA is then sizefractionated by agarose gel electrophoresis, and the fraction includingthe DNA fragment covering the expressed V segment is cloned into lambdaEMBL3 or Lambda FIX (Stratagene, La Jolla, Calif.). V segment containingclones are isolated using the unique probe o-kappa. DNA is isolated frompositive clones and subcloned into the polylinker of pKap1. Theresulting clone is called pRKL.

EXAMPLE 7 Isolation of Genomic Clones Corresponding to RearrangedExpressed Copies of Immunoglobulin Heavy Chain μ Genes

This example describes the cloning of immunoglobulin heavy chain μ genesfrom cultured cells of expressed and immunoglobulin of interest. Theprocedure described in this example allows for the selective cloning ofthe expressed copy of a μ heavy chain gene.

Double-stranded cDNA is prepared and isolated as described hereinbefore. The double-stranded cDNA is denatured and used as a template fora third round of DNA synthesis using the following oligonucleotideprimer: ##STR15##

This primer contains sequences specific for the constant portion of theμ heavy chain message (ACA GGA GAC GAG GGG GAA AAG GGT TGG GGC GGA TGC)as well as unique sequences that can be used as a primer for the PCRamplification of the newly synthesized DNA strand (GTA CGC CAT ATC AGCTGG ATG AAG). The sequence is amplified by PCR using the following twooligonucleotide primers: ##STR16##

The PCR amplified sequence is then purified by gel electrophoresis andused as template for dideoxy sequencing reactions using the followingoligonucleotide as a primer: ##STR17##

The first 42 nucleotides of sequence are then used to synthesize aunique probe for isolating the gene from which immunoglobulin messagewas transcribed. This synthetic 42 nucleotide segment of DNA will bereferred to below as o-mu.

A Southern blot of DNA, isolated from the Ig expressing cell line anddigested individually and in pairwise combinations with severaldifferent restriction endonucleases including MluI (MluI is a rarecutting enzyme that cleaves between the J segment and mu CH1), is thenprobed with the 32-P labelled unique oligonucleotide o-mu. A uniquerestriction endonuclease site is identified upstream of the rearranged Vsegment.

DNA from the Ig expressing cell line is then cut with MluI and secondenzyme. MluI or SpeI adapter linkers are then ligated onto the ends andcut to convert the upstream site to MluI or SpeI. The DNA is then sizefractionated by agarose gel electrophoresis, and the fraction includingthe DNA fragment covering the expressed V segment is cloned directlyinto the plasmid pGPI. V segment containing clones are isolated usingthe unique probe o-mu, and the insert is subcloned into M1uI orM1uI/SpeI cut plasmid pCON2. The resulting plasmid is called pRMGH.

EXAMPLE 8 Construction of Human κ Miniloci Transgenes Light ChainMinilocus

A human genomic DNA phage library was screened with kappa light chainspecific oligonucleotide probes and isolated clones spanning the J.sub.κ-C region. A 5.7 kb C1aI/XhoI fragment containing J.sub.κ 1 togetherwith a 13 kb XhoI fragment containing J_(K) 2-5 and C.sub.κ into pGP1dwas cloned and used to create the plasmid pKcor. This plasmid containsJ.sub.κ 1-5, the kappa intronic enhancer and C.sub.κ together with 4.5kb of 5' and 9 kb of 3' flanking sequences. It also has a unique 5' XhoIsite for cloning V.sub.κ segments and a unique 3' SalI site forinserting additional cis-acting regulatory sequences.

V kappa qenes

A human genomic DNA phage library was screened with V.sub.κ light chainspecific oligonucleotide probes and isolated clones containing humanV.sub.κ segments. Functional V segments were identified by DNA sequenceanalysis. These clones contain TATA boxes, open reading frames encodingleader and variable peptides (including 2 cysteine residues), splicesequences, and recombination heptamer-12 bp spacer-nonamer sequences.Three of the clones were mapped and sequenced. Two of the clones, 65.5and 65.8 appear to be functional, they contain TATA boxes, open readingframes encoding leader and variable peptides (including 2 cysteineresidues), splice sequences, and recombination heptamer-12 bpspacer-nonamer sequences. The third clone, 65.4, appears to encode aV.sub.κ I pseudogene as it contains a non-canonical recombinationheptamer.

One of the functional clones, Vk 65-8, which encodes a VkIII familygene, was used to build a light chain minilocus construct.

pKC1

The kappa light chain minilocus transgene pKC1 (FIG. 32) was generatedby inserting a 7.5 kb XhoI/Sa1I fragment containing V.sub.κ 65.8 intothe 5' XhoI site of pKcor. The transgene insert was isolated bydigestion with NotI prior to injection.

The purified insert was microinjected into the pronuclei of fertilized(C57BL/6×CBA)F2 mouse embryos and transferred the surviving embryos intopseudopregnant females as described by Hogan et al. (in Methods ofManipulating the Mouse Embryo, 1986, Cold Spring Harbor Laboratory, NewYork). Mice that developed from injected embryos were analyzed for thepresence of transgene sequences by Southern blot analysis of tail DNA.Transgene copy number was estimated by band intensity relative tocontrol standards containing known quantities of cloned DNA. Serum wasisolated from these animals and assayed for the presence of transgeneencoded human Ig kappa protein by ELISA as described by Harlow and Lane(in Antibodies: A Laboratory Manual, 1988, Cold Spring HarborLaboratory, New York). Microtiter plate wells were coated with mousemonoclonal antibodies specific for human Ig kappa (clone 6E1, #0173,AMAC, Inc., Westbrook, Me.), human IgM (Clone AF6, #0285, AMAC, Inc.,Westbrook, Me.) and human IgG1 (clone JL512, #0280, AMAC, Inc.,Westbrook, Me.). Serum samples were serially diluted into the wells andthe presence of specific immunoglobulins detected with affinity isolatedalkaline phosphatase conjugated goat anti-human Ig (polyvalent) that hadbeen pre-adsorbed to minimize cross-reactivity with mouseimmunoglobulins.

FIG. 35 shows the results of an ELISA assay of serum from 8 mice (I.D.#676, 674, 673, 670, 666, 665, 664, and 496). The first seven of thesemice developed from embryos that were injected with the pKC1 transgeneinsert and the eighth mouse is derived from a mouse generated bymicroinjection of the pHC1 transgene (described previously). Two of theseven mice from KC1 injected embryos (I.D.#'s 666 and 664) did notcontain the transgene insert as assayed by DAN Southern blot analysis,and five of the mice (I.D.#'s 676, 674, 673, 670, and 665) contained thetransgene. All but one of the KC1 transgene positive animals expressdetectable levels of human Ig kappa protein, and the singlenon-expressing animal appears to be a genetic mosaic on the basis of DNASouthern blot analysis. The pHC1 positive transgenic mouse expresseshuman IgM and IgG1 but not Ig kappa, demonstrating the specificity ofthe reagents used in the assay.

pKC2

The kappa light chain minilocus transgene pKC2 was generated byinserting an 8 kb XhoI/Sa1I fragment containing V_(K) 65.5 into the 5'XhoI site of pKC1. The resulting transgene insert, which contains twoV.sub.κ segments, was isolated prior to microinjection by digestion withNotI.

pKVe2

This construct is identical to pKC1 except that it includes 1.2 kb ofadditional sequence 5' of J.sub.κ and is missing 4.5 kb of sequence 3'of V.sub.κ 65.8. In additional it contains a 0.9 kb XbaI fragmentcontaining the mouse heavy chain J-m intronic enhancer (Banerji et al.,Cell 33:729-740 (1983)) together with a 1.4 kb MluI HindIII fragmentcontaining the human heavy chain J-m intronic enhancer (Hayday et al.,Nature 307:334-340 (1984)) inserted downstream. This construct tests thefeasibility of initiating early rearrangement of the light chainminilocus to effect allelic and isotypic exclusion. Analogous constructscan be generated with different enhancers, i.e., the mouse or rat 3'kappa or heavy chain enhancer (Meyer and Neuberger, EMBO J. 8:1959-1964(1989); Petterson et al. Nature 344:165-168 (1990), which areincorporated herein by reference).

Rearranged Light Chain Transgenes

A kappa light chain expression cassette was designed to reconstructfunctionally rearranged light chain genes that have been amplified byPCR from human B-cell DNA. The scheme is outlined in FIG. 33. PCRamplified light chain genes are cloned into the vector pK5nx thatincludes 3.7 kb of 5' flanking sequences isolated from the kappa lightchain gene 65.5. The VJ segment fused to the 5' transcriptionalsequences are then cloned into the unique XhoI site of the vector pK31sthat includes J.sub.κ 2-4, the J.sub.κ intronic enhancer, C_(K), and 9kb of downstream sequences. The resulting plasmid contains areconstructed functionally rearranged kappa light chain transgene thatcan be excised with NotI for microinjection into embryos. The plasmidsalso contain unique Sa1I sites at the 3' end for the insertion ofadditional ci-sacting regulatory sequences.

Two synthetic oligonucleotides (o-130, o-131) were used to amplifyrearranged kappa light chain genes from human spleen genomic DNA.Oligonucleotide o-131 (gga ccc aga (g,c)gg aac cat gga a(g,a)(g,a,t,c))is complementary to the 5' region of V.sub.κ III family light chaingenes and overlaps the first ATC of the leader sequence. Oligonucleotideo-130 (gtg caa tca att ctc gag ttt gac tac aga c) is complementary to asequence approximately 150 bp 3' of J.sub.κ 1 and includes an XhoI site.These two oligonucleotides amplify a 0.7 kb DNA fragment from humanspleen DNA corresponding to rearranged V.sub.κ III genes joined toJ.sub.κ 1 segments. The PCR amplified DNA was digested with NcoI andXhoI and cloned individual PCR products into the plasmid pNN03. The DNAsequence of 5 clones was determined and identified two with functionalVJ joints (open reading frames). Additional functionally rearrangedlight chain clones are collected. The functionally rearranged clones canbe individually cloned into light chain expression cassette describedabove (FIG. 33). Transgenic mice generated with the rearranged lightchain constructs can be bred with heavy chain minilocus transgenics toproduce a strain of mice that express a spectrum of fully humanantibodies in which all of the diversity of the primary repertoire iscontributed by the heavy chain. One source of light chain diversity canbe from somatic mutation. Because not all light chains will beequivalent with respect to their ability to combine with a variety ofdifferent heavy chains, different strains of mice, each containingdifferent light chain constructs can be generated and tested. Theadvantage of this scheme, as opposed to the use of unrearranged lightchain miniloci, is the increased light chain allelic and isotypicexclusion that comes from having the light chain ready to pair with aheavy chain as soon as heavy chain VDJ joining occurs. This combinationcan result in an increased frequency of B-cells expressing fully humanantibodies, and thus it can facilitate the isolation of human Igexpressing hybridomas.

NotI inserts of plasmids pIGM1, pHC1, pIGG1, pKC1, and pKC2 wereisolated away from vector sequences by agarose gel electrophoresis. Thepurified inserts were microinjected into the pronuclei of fertilized(C57BL/6×CBA) F2 mouse embryos and transferred the surviving embryosinto pseudopregnant females as described by Hogan et al. (Hogan et al.,Methods of Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory,New York (1986)).

EXAMPLE 9 Inactivation of the Mouse Kappa Liqht Chain Gene by HomologousRecombination

This example describes the inactivation of the mouse endogenous kappalocus by homologous recombination in embryonic stem (ES) cells followedby introduction of the mutated gene into the mouse germ line byinjection of targeted ES cells bearing an inactivated kappa allele intoearly mouse embryos (blastocysts).

The strategy is to delete J_(K) and C_(K) by homologous recombinationwith a vector containing DNA sequences homologous to the mouse kappalocus in which a 4.5 kb segment of the locus, spanning the J_(K) geneand C_(K) segments, is deleted and replaced by the selectable markerneo.

Construction of the kappa targeting vector

The plasmid pGEM7 (KJ1) contains the neomycin resistance gene (neo),used for drug selection of transfected ES cells, under thetranscriptional control of the mouse phosphoglycerate kinase (pgk)promoter (XbaI/TaqI fragment; Adra et al., Gene 60:65-74 (1987)) in thecloning vector pGEM7Zf(+). The plasmid also includes a heterologouspolyadenylation site for the neogene, derived from the 3' region of themouse pgk gene (PvuII/HindIII fragment; Boer et al., BiochemicalGenetics, 28:299-308 (1990)). This plasmid was used as the startingpoint for construction of the kappa targeting vector. The first step wasto insert sequences homologous to the kappa locus 3' of the neoexpression cassette.

Mouse kappa chain sequences (FIG. 20a) were isolated from a genomicphage library derived from liver DNA using oligonucleotide probesspecific for the CK locus: ##STR18## and for the Jκ5 gene segment:##STR19##

An 8 kb Bg1II/SacI fragment extending 3' of the mouse C_(K) segment wasisolated from a positive phage clone in two pieces, as a 1.2 kbBg1II/SacI fragment and a 6.8 kb SacI fragment, and subcloned intoBg1II/SacI digested pGEM7 (KJ1) to generate the plasmid pNEO-K3' (FIG.20b).

A 1.2 kb EcoRI/SphI fragment extending 5' of the J_(K) region was alsoisolated from a positive phage clone. An SphI/XbaI/Bg1II/EcoRI adaptorwas ligated to the SphI site of this fragment, and the resulting EcoRIfragment was ligated into EcoRI digested pNEO-K3', in the same 5' to 3'orientation as the neogene and the downstream 3' kappa sequences, togenerate pNEO-K5'3' (FIG. 20c).

The Herpes Simplex Virus (HSV) thymidine kinase (TK) gene was thenincluded in the construct in order to allow for enrichment of ES clonesbearing homologous recombinants, as described by Mansour et al., Nature336:348-352 (1988), which is incorporated herein by reference. The HSVTK cassette was obtained from the plasmid pGEM7 (TK), which contains thestructural sequences for the HSV TK gene bracketed by the mouse pgkpromoter and polyadenylation sequences as described above for pGEM7(KJ1). The EcoRI site of pGEM7 (TK) was modified to a BamHI site and theTK cassette was then excised as a BamHI/HindIII fragment and subclonedinto pGP1b to generate pGP1b-TK. This plasmid was linearized at the XhoIsite and the XhoI fragment from pNEO-K5'3', containing the neo geneflanked by genomic sequences from 5' of Jκ and 3' of Cκ, was insertedinto pGP1b-TK to generate the targeting vector J/C KI (FIG. 20d). Theputative structure of the genomic kappa locus following homologousrecombination with J/C K1 is shown in FIG. 20e.

Generation and analysis of ES cells with targeted inactivation of akappa allele

The ES cells used were the AB-1 line grown on mitotically inactiveSNL76/7 cell feeder layers (McMahon and Bradley, Cell 62:1073-1085(1990)) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J.Robertson, ed. (Oxford: IRL Press), p. 71-112). Other suitable ES linesinclude, but are not limited to, the E14 line (Hooper et al. (1987)Nature 326: 292-295), the D3 line (Doetschman et al. (1985) J. Embryol.Exp. Morph. 87:27-45), and the CCE line (Robertson et al. (1986) Nature323: 445-448). The success of generating a mouse line from ES cellsbearing a specific targeted mutation depends on the pluripotence of theES cells (i.e., their ability, once injected into a host blastocyst, toparticipate in embryogenesis and contribute to the germ cells of theresulting animal).

The pluripotence of any given ES cell line can vary with time in cultureand the care with which it has been handled. The only definitive assayfor pluripotence is to determine whether the specific population of EScells to be used for targeting can give rise to chimeras capable ofgermline transmission of the ES genome. For this reason, prior to genetargeting, a portion of the parental population of AB-1 cells isinjected into C57B1/6J blastocysts to ascertain whether the cells arecapable of generating chimeric mice with extensive ES cell contributionand whether the majority of these chimeras can transmit the ES genome toprogeny.

The kappa chain inactivation vector J/C K1 was digested with NotI andelectroporated into AB-1 cells by the methods described (Hasty et al.,Nature, 350:243-246 (1991)). Electroporated cells were plated onto 100mm dishes at a density of 1-2×106 cells/dish. After 24 hours, G418 (200μg/ml of active component) and FIAU (0.5μM) were added to the medium, anddrug-resistant clones were allowed to develop over 10-11 days. Cloneswere picked, trypsinized, divided into two portions, and furtherexpanded. Half of the cells derived from each clone were then frozen andthe other half analyzed for homologous recombination between vector andtarget sequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described (Laird et al., Nucl. Acids Res.19:4293 (1991)) digested with XbaI and probed with the 800 bp EcoRI/XbaIfragment indicated in FIG. 20e as probe A. This probe detects a 3.7 kbXbaI fragment in the wild type locus, and a diagnostic 1.8 kb band in alocus which has homologously recombined with the targeting vector (seeFIG. 20a and e). Of 901 G418 and FIAU resistant clones screened bySouthern blot analysis, 7 displayed the 1.8 kb XbaI band indicative of ahomologous recombination into one of the kappa genes. These 7 cloneswere further digested with the enzymes Bg1II, SacI, and PstI to verifythat the vector integrated homologously into one of the kappa genes.When probed with the diagnostic 800 bp EcoRI/XbaI fragment (probe A),Bg1II, SacI, and PstI digests of wild type DNA produce fragments of 4.1,5.4, and 7 kb, respectively, whereas the presence of a targeted kappaallele would be indicated by fragments of 2.4, 7.5, and 5.7 kb,respectively (see FIG. 20a and e). All 7 positive clones detected by theXbaI digest showed the expected Bg1II, SacI, and PstI restrictionfragments diagnostic of a homologous recombination at the kappa lightchain. In addition, Southern blot analysis of an NsiI digest of thetargeted clones using a neo specific probe (probe B, FIG. 20e) generatedonly the predicted fragment of 4.2 kb, demonstrating that the cloneseach contained only a single copy of the targeting vector.

Generation of mice bearing the inactivated kappa chain

Five of the targeted ES clones described in the previous section werethawed and injected into C57Bl/6J blastocysts as described (Bradley, A.(1987) in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach. E. J. Robertson, ed. (Oxford: IRL Press), p. 113-151) andtransferred into the uteri of pseudopregnant females to generatechimeric mice resulting from a mixture of cells derived from the inputES cells and the host blastocyst. The extent of ES cell contribution tothe chimeras can be visually estimated by the amount of agouti coatcoloration, derived from the ES cell line, on the black C57Bl/6Jbackground. Approximately half of the offspring resulting fromblastocyst injection of the targeted clones were chimeric (i.e., showedagouti as well as black pigmentation) and of these, the majority showedextensive (70 percent or greater) ES cell contribution to coatpigmentation. The AB1 ES cells are an XY cell line and a majority ofthese high percentage chimeras were male due to sex conversion of femaleembryos colonized by male ES cells. Male chimeras derived from 4 of the5 targeted clones were bred with C57BL/6J females and the offspringmonitored for the presence of the dominant agouti coat color indicativeof germline transmission of the ES genome. Chimeras from two of theseclones consistently generated agouti offspring. Since only one copy ofthe kappa locus was targeted in the injected ES clones, each agouti puphad a 50 percent chance of inheriting the mutated locus. Screening forthe targeted gene was carried out by Southern blot analysis of BglII-digested DNA from tail biopsies, using the probe utilized inidentifying targeted ES clones (probe A, FIG. 20e). As expected,approximately 50 percent of the agouti offspring showed a hybridizingBgl II band of 2.4 kb in addition to the wild-type band of 4.1 kb,demonstrating the germline transmission of the targeted kappa locus.

In order to generate mice homozygous for the mutation, heterozygoteswere bred together and the kappa genotype of the offspring determined asdescribed above. As expected, three genotypes were derived from theheterozygote matings: wild-type mice bearing two copies of a normalkappa locus, heterozygotes carrying one targeted copy of the kappa geneand one NT kappa gene, and mice homozygous for the kappa mutation. Thedeletion of kappa sequences from these latter mice was verified byhybridization of the Southern blots with a probe specific for J_(K)(probe C, FIG. 20a). Whereas hybridization of the J_(K) probe wasobserved to DNA samples from heterozygous and wild-type siblings, nohybridizing signal was present in the homozygotes, attesting to thegeneration of a novel mouse strain in which both copies of the kappalocus have been inactivated by deletion as a result of targetedmutation.

EXAMPLE 10 Inactivation of the Mouse Heavy Chain Gene by HomologousRecombination

This example describes the inactivation of the endogenous murineimmunoglobulin heavy chain locus by homologous recombination inembryonic stem (ES) cells. The strategy is to delete the endogenousheavy chain J segments by homologous recombination with a vectorcontaining heavy chain sequences from which the J_(H) region has beendeleted and replaced by the gene for the selectable marker neo.

Construction of a heavy chain targeting vector

Mouse heavy chain sequences containing the J_(H) region (FIG. 21a) wereisolated from a genomic phage library derived from the D3 ES cell line(Gossler et al., Proc. Natl. Acad. Sci. U.S.A. 83:9065-9069 (1986))using a J_(H) 4 specific oligonucleotide probe: ##STR20##

A 3.5 kb genomic SacI/StuI fragment, spanning the J_(H) region, wasisolated from a positive phage clone and subcloned into SacI/SmaIdigested pUC18. The resulting plasmid was designated pUC18 J_(H). Theneomycin resistance gene (neo), used for drug selection of transfectedES cells, was derived from a repaired version of the plasmid pGEM7(KJ1). A report in the literature (Yenofsky et al. (1990) Proc. Natl.Acad. Sci. (U.S.A.) 87: 3435-3439) documents a point mutation the neocoding sequences of several commonly used expression vectors, includingthe construct pMClneo (Thomas and Cappechi (1987) Cell 51: 503-512)which served as the source of the neogene used in pGEM7 (KJ1). Thismutation reduces the activity of the neogene product and was repaired byreplacing a restriction fragment encompassing the mutation with thecorresponding sequence from a wild-type neo clone. The HindIII site inthe prepared pGEM7 (KJ1) was converted to a SalI site by addition of asynthetic adaptor, and the neo expression cassette excised by digestionwith XbaI/SalI. The ends of the neo fragment were then blunted bytreatment with the Klenow form of DNA polI, and the neo fragment wassubcloned into the NaeI site of pUC18 J_(H), generating the plasmidpUC18 J_(H) -neo (FIG. 21b).

Further construction of the targeting vector was carried out in aderivative of the plasmid pGP1b. pGP1b was digested with the restrictionenzyme NotI and ligated with the following oligonucleotide as anadaptor: ##STR21##

The resulting plasmid, called pGMT, was used to build the mouseimmunoglobulin heavy chain targeting construct.

The Herpes Simplex Virus (HSV) thymidine kinase (TK) gene was includedin the construct in order to allow for enrichment of ES clones bearinghomologous recombinants, as described by Mansour et al. (Nature 336,348-352 (1988)). The HSV TK gene was obtained from the plasmid pGEM7(TK) by digestion with EcoRI and HindIII. The TK DNA fragment wassubcloned between the EcoRI and HindIII sites of pGMT, creating theplasmid pGMT-TK (FIG. 21c).

To provide an extensive region of homology to the target sequence, a 5.9kb genomic XbaI/XhoI fragment, situated 5' of the J_(H) region, wasderived from a positive genomic phage clone by limit digestion of theDNA with XhoI, and partial digestion with XbaI. As noted in FIG. 21a,this XbaI site is not present in genomic DNA, but is rather derived fromphage sequences immediately flanking the cloned genomic heavy chaininsert in the positive phage clone. The fragment was subcloned intoXbaI/XhoI digested pGMT-TK, to generate the plasmid pGMT-TK-J_(H) 5'(FIG. 21d).

The final step in the construction involved the excision from pUC18J_(H) -neo of the 2.8 kb EcoRI fragment which contained the neogene andflanking genomic sequences 3' of J_(H). This fragment was blunted byKlenow polymerase and subcloned into the similarly blunted XhoI site ofpGMT-TK-J_(H) 5'. The resulting construct, J_(H) KO1 (FIG. 21e),contains 6.9 kb of genomic sequences flanking the J_(H) locus, with a2.3 kb deletion spanning the J_(H) region into which has been insertedthe neogene. FIG. 21f shows the structure of an endogenous heavy chaingene after homologous recombination with the targeting construct.

EXAMPLE 11 Generation and analysis of targeted ES cells

AB-1 ES cells (McMahon and Bradley, Cell 62:1073-1085 (1990)) were grownon mitotically inactive SNL76/7 cell feeder layers essentially asdescribed (Robertson, E. J. (1987) Teratocarcinomas and Embryonic StemCells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press),pp. 71-112). As described in the previous example, prior toelectroporation of ES cells with the targeting construc J_(H) KO1, thepluripotency of the ES cells was determined by generation of AB-1derived chimeras which were shown capable of germline transmission ofthe ES genome.

The heavy chain inactivation vector J_(H) KO1 was digested with NotI andelectroporated into AB-1 cells by the methods described (Hasty et al.,Nature 350:243-246 (1991)). Electroporated cells were plated into 100 mmdishes at a density of 1-2×10⁶ cells/dish. After 24 hours, G418 (200mg/ml of active component) and FIAU (0.5 mM) were added to the medium,and drug-resistant clones were allowed to develop over 8-10 days. Cloneswere picked, trypsinized, divided into two portions, and furtherexpanded. Half of the cells derived from each clone were then frozen andthe other half analyzed for homologous recombination between vector andtarget sequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described (Laird et al. (1991) Nucleic AcidsRes. 19: 4293), digested with StuI and probed with the 500 bp EcoRI/StuIfragment designated as probe A in FIG. 21f. This probe detects a StuIfragment of 4.7 kb in the wild-type locus, whereas a 3 kb band isdiagnostic of homologous recombination of endogenous sequences with thetargeting vector (see FIG. 21a and f). Of 525 G418 and FIAUdoubly-resistant clones screened by Southern blot hybridization, 12 werefound to contain the 3 kb fragment diagnostic of recombination with thetargeting vector. That these clones represent the expected targetedevents at the J_(H) locus (as shown in FIG. 21f) was confirmed byfurther digestion with HindIII, SpeI and HpaI. Hybridization of probe A(see FIG. 21f) to Southern blots of HindIII, SpeI, and HpaI digested DNAproduces bands of 2.3 kb, >10 kb, and >10 kb, respectively, for thewild-type locus (see FIG. 21a), whereas bands of 5.3 kb, 3.8 kb, and 1.9kb, respectively, are expected for the targeted heavy chain locus (seeFIG. 21f). All 12 positive clones detected by the StuI digest showed thepredicted HindIII, SpeI, and HpaI bands diagnostic of a targeted J_(H)gene. In addition, Southern blot analysis of a StuI digest of all 12clones using a neo-specific probe (probe B, FIG. 21f) generated only thepredicted fragment of 3 kb, demonstrating that the clones each containedonly a single copy of the targeting vector.

Generation of mice carrying the J_(H) deletion

Three of the targeted ES clones described in the previous section werethawed and injected into C57BL/6J blastocysts as described (Bradley, A.(1987) in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (Oxford: IRL Press), p. 113-151) andtransferred into the uteri of pseudopregnant females. The extent of EScell contribution to the chimera was visually estimated from the amountof agouti coat coloration, derived from the ES cell line, on the blackC57BL/6J background. Half of the offspring resulting from blastocystinjection of two of the targeted clones were chimeric (i.e., showedagouti as well as black pigmentation); the third targeted clone did notgenerate any chimeric animals. The majority of the chimeras showedsignificant (approximately 50 percent or greater) ES cell contributionto coat pigmentation. Since the AB-1 ES cells are an XY cell line, mostof the chimeras were male, due to sex conversion of female embryoscolonized by male ES cells. Males chimeras were bred with C57BL/6Jfemales and the offspring monitored for the presence of the dominantagouti coat color indicative of germline transmission of the ES genome.Chimeras from both of the clones consistently generated agoutioffspring. Since only one copy of the heavy chain locus was targeted inthe injected ES clones, each agouti pup had a 50 percent chance ofinheriting the mutated locus. Screening for the targeted gene wascarried out by Southern blot analysis of StuI-digested DNA from tailbiopsies, using the probe utilized in identifying targeted ES clones(probe A, FIG. 21f). As expected, approximately 50 percent of the agoutioffspring showed a hybridizing StuI band of approximately 3 kb inaddition to the wild-type band of 4.7 kb, demonstrating germlinetransmission of the targeted J_(H) gene segment.

In order to generate mice homozygous for the mutation, heterozygoteswere bred together and the heavy chain genotype of the offspringdetermined as described above. As expected, three genotypes were derivedfrom the heterozygote matings: wild-type mice bearing two copies of thenormal J_(H) locus, heterozygotes caring one targeted copy of the geneand one normal copy, and mice homozygous for the J_(H) mutation. Theabsence of J_(H) sequences from these latter mice was verified byhybridization of the Southern blots of StuI-digested DNA with a probespecific for J_(H) (probe C, FIG. 21a). Whereas hybridization of theJ_(H) probe to a 4.7 kb fragment in DNA samples from heterozygous andwild-type siblings was observed, no signal was present in samples fromthe J_(H) -mutant homozygotes, attesting to the generation of a novelmouse strain in which both copies of the heavy chain gene have beenmutated by deletion of the J_(H) sequences.

EXAMPLE 12 Heavy Chain Minilocus Transgene

A. Construction of plasmid vectors for cloning large DNA sequences

1. pGP1a

The plasmid pBR322 was digested with EcoRI and StyI and ligated with thefollowing oligonucleotides: ##STR22##

The resulting plasmid, pGP1a, is designed for cloning very large DNAconstructs that can be excised by the rare cutting restriction enzymeNotI. It contains a NotI restriction site downstream (relative to theampicillin resistance gene, AmpR) of a strong transcription terminationsignal derived from the trpA gene (Christie et al., Proc. Natl. Acad.Sci. USA 78:4180 (1981)). This termination signal reduces the potentialtoxicity of coding sequences inserted into the NotI site by eliminatingreadthrough transcription from the AmpR gene. In addition, this plasmidis low copy relative to the pUC plasmids because it retains the pBR322copy number control region. The low copy number further reduces thepotential toxicity of insert sequences and reduces the selection againstlarge inserts due to DNA replication. The vectors pGP1b, pGP1c, pGP1d,and pGP1f are derived from pGP1a and contain different polylinkercloning sites. The polylinker sequences are given below ##STR23## Eachof these plasmids can be used for the construction of large transgeneinserts that are excisable with NotI so that the transgene DNA can bepurified away from vector sequences prior to microinjection.

2. pGP1b

pGP1a was digested with NotI and ligated with the followingoligonucleotides: ##STR24##

The resulting plasmid, pGP1b, contains a short polylinker region flankedby NotI sites. This facilitates the construction of large inserts thatcan be excised by NotI digestion.

3. pGPe

The following oligonucleotides: ##STR25## were used to amplify theimmunoglobulin heavy chain 3' enhancer (S. Petterson, et al., Nature344:165-168 (1990)) from rat liver DNA by the polymerase chain reactiontechnique.

The amplified product was digested with BamHI and SphI and cloned intoBamHI/SphI digested pNNO3 (pNNO3 is a pUC derived plasmid that containsa polylinker with the following restriction sites, listed in order:NotI, BamHI, NcoI, ClaI, EcoRV, XbaI, SacI, XhoI, SphI, PstI, BglII,EcoRI, SmaI, KpnI, HindIII, and NotI). The resulting plasmid, pRE3, wasdigested with BamHI and HindIII, and the insert containing the rat Igheavy chain 3' enhancer cloned into BamHI/HindIII digested pGP1b. Theresulting plasmid, pGPe (FIG. 22 and Table 1), contains several uniquerestriction sites into which sequences can be cloned and subsequentlyexcised together with the 3' enhancer by NotI digestion.

                                      TABLE 1                                     __________________________________________________________________________    AATTAGCggccgcctcgagatcactatcgattaattaaggatccagatatcagtacctgaaacagggcttgctc    c                                                                             tctctctctctgtctctctgtctctgtgtgtgtgtctctctctgtctctgtctctctctgtctctctgtctctg    g                                                                             tctctctctgtctctctctctgtctctctgtctctctgtctgtctctgtctctgtctctgtctctctctctctc    c                                                                             tctctctctctctctctcacacacacacacacacacacacacacacctgccgagtgactcactctgtgcagggt    g                                                                             tcggggcacatgcaaatggatgtttgttccatgcagaaaaacatgtttctcattctctgagccaaaaatagcat    a                                                                             ttcccccaccctgcagctgcaggttcaccccacctggccaggttgaccagctttggggatggggctgggggttc    a                                                                             ccctaacggtgacattgaattcagtgttttcccatttatcgacactgctggaatctgaccctaggagggaatga    a                                                                             ataggcaaggtccaaacaccccagggaagtgggagagacaggaaggctgtgtgtgctccaggtcctgtgcatgc    g                                                                             tctgaattcccgggtaccaagcttgcGGCCGCAGTATGCAAAAAAAAGCCCGCTCATTAGGCGGGCTCTTGGCA    GAACAT                                                                        ATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGC    CAGAT                                                                         TCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATA    CGCGAG                                                                        CGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGT    TA                                                                            AAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTAC    TG                                                                            GAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCAT    CCGCC                                                                         AGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTC    TTT                                                                           CATCGGTATCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGGCATCAAGTGACCAAACAGGAAAAAAC    CCCT                                                                          TAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAAC    GGCAG                                                                         ACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGT    AAAAC                                                                         CTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCA    GGGCGC                                                                        GTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCT    ACTA                                                                          TGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAA    AATACC                                                                        GCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA    GCTCAC                                                                        TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATTAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGC    AAAGGC                                                                        CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCAGAAAAATC    ACGCT                                                                         CAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC    C                                                                             GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTC    CGCTG                                                                         TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACC    CTGCG                                                                         CCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGT    ACAGG                                                                         ATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAG    GACAGT                                                                        ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA    CCACCG                                                                        CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG    ATCTTT                                                                        TCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGAT    CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTT    CCAAT                                                                         GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG    TAGATA                                                                        ACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCC    GATTT                                                                         ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGT    TTA                                                                           ATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGC    TCGTG                                                                         GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC    TGTT                                                                          GTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCA    TGGTTA                                                                        TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC    AGTCA                                                                         TTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAG    CAGAAC                                                                        TTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA    GTTCGA                                                                        TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA    GGAAGG                                                                        CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA    TTGAAG                                                                        CATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC    CGCA                                                                          CATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGT    TCACG                                                                         AGGCCCTTTCGTCTTCAAG                                                           Sequence of vector pGPe.                                                      __________________________________________________________________________

B. Construction of IgM expressing minilocus transgene, pIGM1

1. Isolation of J-μ constant region clones and construction of pJM1

A human placental genomic DNA library cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) wasscreened with the human heavy chain J region specific oligonucleotide:##STR26## and the phage clone λ1.3 isolated. A 6 kb HindIII/KpnIfragment from this clone, containing all six J segments as well as Dsegment DHQ52 and the heavy chain J-μ intronic enhancer, was isolated.The same library was screened with the human μ specific oligonucleotide:##STR27## and the phage clone λ2.1 isolated. A 10.5 kb HindIII/XhoIfragment, containing the μ switch region and all of the μ constantregion exons, was isolated from this clone. These two fragments wereligated together with KpnI/XhoI digested pNNO3 to obtain the plasmidpJM1. 2. pJM2

A 4 kb XhoI fragment was isolated from phage clone λ2.1 that containssequences immediately downstream of the sequences in pJM1, including theso called Σμ element involved in δ-associated deleteon of the μ incertain IgD expressing B-cells (Yasui et al., Eur. J. Immunol. 19:1399(1989), which is incorporated herein by reference). This fragment wastreated with the Klenow fragment of DNA polymerase I and ligated to XhoIcut, Klenow treated, pJM1. The resulting plasmid, pJM2 (FIG. 23), hadlost the internal XhoI site but retained the 3' XhoI site due toincomplete reaction by the Klenow enzyme. pJM2 contains the entire humanJ region, the heavy chain J-μ intronic enhancer, the μ switch region andall of the μ constant region exons, as well as the two 0.4 kb directrepeats, σμ and Σμ, involved in δ-associated deletion of the μ gene.

3. Isolation of D region clones and construction of pDH1

The following human D region specific oligonucleotide: ##STR28## wasused to screen the human placenta genomic library for D region clones.Phage clones λ4.1 and λ4.3 were isolated. A 5.5 kb XhoI fragment, thatincludes the D elements D_(K1), D_(N1), and D_(M2) (Ichihara et al.,EMBO J. 7:4141 (1988)), was isolated from phage clone λ4.1. An adjacentupstream 5.2 kb XhoI fragment, that includes the D elements D_(LR1),D_(Xp1), D_(xp'1), and D_(A1), was isolated from phage clone λ4.3. Eachof these D region XhoI fragments were cloned into the SalI site of theplasmid vector pSP72 (Promega, Madison, Wis.) so as to destroy the XhoIsite linking the two sequences. The upstream fragment was then excisedwith XhoI and SmaI, and the downstream fragment with EcoRV and XhoI. Theresulting isolated fragments were ligated together with SalI digestedpSP72 to give the plasmid pDH1. pDH1 contains a 10.6 kb insert thatincludes at least 7 D segments and can be excised with XhoI (5') andEcoRV (3').

4. pCOR1

The plasmid pJM2 was digested with Asp718 (an isoschizomer of KpnI) andthe overhang filled in with the Klenow fragment of DNA polymerase I. Theresulting DNA was then digested with ClaI and the insert isolated. Thisinsert was ligated to the XhoI/EcoRV insert of pDH1 and XhoI/ClaIdigested pGPe to generate pCOR1 (FIG. 24).

5. pVH251

A 10.3 kb genomic HindIII fragment containing the two human heavy chainvariable region segments V_(H) 251 and V_(H) 105 (Humphries et al.,Nature 331:446 (1988), which is incorporated herein by reference) wassubcloned into pSP72 to give the plasmid pVH251.

6. pIGM1

The plasmid pCOR1 was partially digested with XhoI and the isolatedXhoI/SalI insert of pVH251 cloned into the upstream XhoI site togenerate the plasmid pIGM1 (FIG. 25). pIGM1 contains 2 functional humanvariable region segments, at least 8 human D segments all 6 human J_(H)segments, the human J-μ enhancer, the human σμ element, the human μswitch region, all of the human μ coding exons, and the human Σμelement, together with the rat heavy chain 3' enhancer, such that all ofthese sequence elements can be isolated on a single fragment, away fromvector sequences, by digestion with NotI and microinjected into mouseembryo pronuclei to generate transgenic animals.

C. Construction of IgM and IgG expressing minilocus transgene, pHC1

1. Isolation of Υ constant reqion clones

The following oligonucleotide, specific for human Ig g constant regiongenes: ##STR29## was used to screen the human genomic library. Phageclones 129.4 and λ29.5 were isolated. A 4 kb HindIII fragment of phageclone λ29.4, containing a Υ switch region, was used to probe a humanplacenta genomic DNA library cloned into the phage vector lambda FIX™ II(Stratagene, La Jolla, Calif.). Phage clone λSg1.13 was isolated. Todetermine the subclass of the different Υ clones, dideoxy sequencingreactions were carried out using subclones of each of the three phageclones as templates and the following oligonucleotide as a primer:##STR30##

Phage clones λ29.5 and λSΥ1.13 were both determined to be of the Υ1subclass.

2. pΥel

A 7.8 kb HindIII fragment of phage clone λ29.5, containing the Υ1 codingregion was cloned into pUC18. The resulting plasmid, pLT1, was digestedwith XhoI, Klenow treated, and religated to destroy the internal XhoIsite. The resulting clone, pLTlxk, was digested with HindIII and theinsert isolated and cloned into pSP72 to generate the plasmid clonepLT1xks. Digestion of pLT1xks at a polylinker XhoI site and a humansequence derived BamHI site generates a 7.6 kb fragment containing theΥ1 constant region coding exons. This 7.6 kb XhoI/BamHI fragment wascloned together with an adjacent downstream 4.5 kb BamHI fragment fromphage clone λ29.5 into XhoI/BamHI digested pGPe to generate the plasmidclone pΥel. pΥel contains all of the Υ1 constant region coding exons,together with 5 kb of downstream sequences, linked to the rat heavychain 3' enhancer.

3. pΥe2

A 5.3 kb HindIII fragment containing the Υ1 switch region and the firstexon of the pre-switch sterile transcript (P. Sideras et al. (1989)International Immunol. 1, 631) was isolated from phage clone λSΥ1.13 andcloned into pSP72 with the polylinker XhoI site adjacent to the 5' endof the insert, to generate the plasmid clone pSΥ1s. The XhoI/SalI insertof pSΥ1s was cloned into XhoI digested pΥel to generate the plasmidclone pΥe2 (FIG. 26). pΥe2 contains all of the Υ1 constant region codingexons, and the upstream switch region and sterile transcript exons,together with 5 kb of downstream sequences, linked to the rat heavychain 3' enhancer. This clone contains a unique XhoI site at the 5' endof the insert. The entire insert, together with the XhoI site and the 3'rat enhancer can be excised from vector sequences by digestion withNotI.

4. pHC1

The plasmid pIGM1 was digested with XhoI and the 43 kb insert isolatedand cloned into XhoI digested pge2 to generate the plasmid pHC1 (FIG.25). pHC1 contains 2 functional human variable region segments, at least8 human D segments all 6 human J_(H) segments, the human J-μ enhancer,the human σμ element, the human μswitch region, all of the human μcoding exons, the human Σμ element, and the human Υ1 constant region,including the associated switch region and sterile transcript associatedexons, together with the rat heavy chain 3' enhancer, such that all ofthese sequence elements can be isolated on a single fragment, away fromvector sequences, by digestion with NotI and microinjected into mouseembryo pronuclei to generate transgenic animals.

D. Construction of IgM and IgG expressing minilocus transgene, pHC2

1. Isolation of human heavy chain V region gene VH49.8

The human placental genomic DNA library lambda, FIX™ II, Stratagene, LaJolla, Calif.) was screened with the following human VH1 family specificoligonucleotide: ##STR31##

Phage clone λ49.8 was isolated and a 6.1 kb XbaI fragment containing thevariable segment VH49.8 subcloned into pNNO3 (such that the polylinkerClaI site is downstream of VH49.8 and the polylinker XhoI site isupstream) to generate the plasmid pVH49.8. An 800 bp region of thisinsert was sequenced, and VH49.8 found to have an open reading frame andintact splicing and recombination signals, thus indicating that the geneis functional (Table 2).

                                      TABLE 2                                     __________________________________________________________________________    TTCCTCAGGCAGGATTTAGGGCTTGGTCTCTCAGCATCCCACACTTGTAC 50                         AGCTGATGTGGCATCTGTGTTTTCTTTCTCATCCTAGATCAAGCTTTGAG100                         CTGTGAAATACCCTGCCTCATGAATATGCAAATAATCTGAGGTCTTCTGA150                         GATAAATATAGATATATTGGTGCCCTGAGAGCATCACATAACAACCAGAT200                          ##STR32##                                                                     ##STR33##                                                                    agtcctaaggctgaggaagggatcctggtttagttaaagaggattttatt350                          ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                                    AGATGACAGGGTTTATTAGGTTTAAGGCTGTTTACAAAATGGGTTATATA800                         TTTGAGAAAAAA812                                                               Sequence of human V.sub.H I family gene V.sub.H 49.8                          __________________________________________________________________________

2. pV2

A 4 kb XbaI genomic fragment containing the human V_(H) IV family geneV_(H) 4-21 (Sanz et al., EMBO J., 8:3741 (1989)), subcloned into theplasmid pUC12, was excised with SmaI and HindIII, and treated with theKlenow fragment of polymerase I. The blunt ended fragment was thencloned into ClaI digested, Klenow treated, pVH49.8. The resultingplasmid, pV2, contains the human heavy chain gene VH49.8 linked upstreamof VH4-21 in the same orientation, with a unique SalI site at the 3' endof the insert and a unique XhoI site at the 5' end.

3. pSΥ1-5'

A 0.7 kb XbaI/HindIII fragment (representing sequences immediatelyupstream of, and adjacent to, the 5.3 kb Υ1 switch region containingfragment in the plasmid pΥe2) together with the neighboring upstream 3.1kb XbaI fragment were isolated from the phage clone λSg1.13 and clonedinto HindIII/XbaI digested pUC18 vector. The resulting plasmid, pSΥ1-5',contains a 3.8 kb insert representing sequences upstream of theinitiation site of the sterile transcript found in B-cells prior toswitching to the Υ1 isotype (P. Sideras et al., International Immunol.1:631 (1989)). Because the transcript is implicated in the initiation ofisotype switching, and upstream cis-acting sequences are often importantfor transcription regulation, these sequences are included in transgeneconstructs to promote correct expression of the sterile transcript andthe associated switch recombination.

4. pVGE1

The pSΥ1-5' insert was excised with SmaI and HindIII, treated withKlenow enzyme, and ligated with the following oligonucleotide linker:##STR42## The ligation product was digested with SalI and ligated toSalI digested pV2. The resulting plasmid, pVP, contains 3.8 kb of Υ1switch 5' flanking sequences linked downstream of the two human variablegene segments VH49.8 and VH4-21 (see Table 2). The pVP insert isisolated by partial digestion with SalI and complete digestion withXhoI, followed by purification of the 15 kb fragment on an agarose gel.The insert is then cloned into the XhoI site of pΥe2 to generate theplasmid clone pVGE1 (FIG. 27). pVGE1 contains two human heavy chainvariable gene segments upstream of the human Υ1 constant gene andassociated switch region. A unique SalI site between the variable andconstant regions can be used to clone in D, J, and μ gene segments. Therat heavy chain 3' enhancer is linked to the 3' end of the Υ1 gene andthe entire insert is flanked by NotI sites.

5. pHC2

The plasmid clone pVGE1 is digested with SalI and the XhoI insert ofpIGM1 is cloned into it. The resulting clone, pHC2 (FIG. 25), contains 4functional human variable region segments, at least 8 human D segmentsall 6 human J_(H) segments, the human J-m enhancer, the human σμelement, the human μ switch region, all of the human μ coding exons, thehuman Σμ element, and the human Υ1 constant region, including theassociated switch region and sterile transcript associated exons,together with 4 kb flanking sequences upstream of the sterile transcriptinitiation site. These human sequences are linked to the rat heavy chain3' enhancer, such that all of the sequence elements can be isolated on asingle fragment, away from vector sequences, by digestion with NotI andmicroinjected into mouse embryo pronuclei to generate transgenicanimals. A unique XhoI site at the 5' end of the insert can be used toclone in additional human variable gene segments to further expand therecombinational diversity of this heavy chain minilocus.

E. Transgenic mice

The NotI inserts of plasmids pIGM1 and pHC1 were isolated from vectorsequences by agarose gel electrophoresis. The purified inserts weremicroinjected into the pronuclei of fertilized (C57BL/6x CBA) F2 mouseembryos and transferred the surviving embryos into pseudopregnantfemales as described by Hogan et al. (B. Hogan, F. Costantini, and E.Lacy, Methods of Manipulating the Mouse Embryo, 1986, Cold Spring HarborLaboratory, New York). Mice that developed from injected embryos wereanalyzed for the presence of transgene sequences by Southern blotanalysis of tail DNA. Transgene copy number was estimated by bandintensity relative to control standards containing known quantities ofcloned DNA. At 3 to 8 weeks of age, serum was isolated from theseanimals and assayed for the presence of transgene encoded human IgM andIgG1 by ELISA as described by Harlow and Lane (E. Harlow and D. Lane.Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory,New York). Microtiter plate wells were coated with mouse monoclonalantibodies specific for human IgM (clone AF6, #0285, AMAC, Inc.Westbrook, Me.) and human IgG1 (clone JL512, #0280, AMAC, Inc.Westbrook, Me.). Serum samples were serially diluted into the wells andthe presence of specific immunoglobulins detected with affinity isolatedalkaline phosphatase conjugated goat anti-human Ig (polyvalent) that hadbeen pre-adsorbed to minimize cross-reactivity with mouseimmunoglobulins. Table 3 and FIG. 28 show the results of an ELISA assayfor the presence of human IgM and IgG1 in the serum of two animals thatdeveloped from embryos injected with the transgene insert of plasmidpHC1. All of the control non-transgenic mice tested negative forexpression of human IgM and IgG1 by this assay. Mice from two linescontaining the pIGM1 NotI insert (lines #6 and 15) express human IgM butnot human IgG1. We tested mice from 6 lines that contain the pHC1 insertand found that 4 of the lines (lines #26, 38, 57 and 122) express bothhuman IgM and human IgG1, while mice from two of the lines (lines #19and 21) do not express detectable levels of human immunoglobulins. ThepHC1 transgenic mice that did not express human immunoglobulins wereso-called G_(o) mice that developed directly from microinjected embryosand may have been mosaic for the presence of the transgene. Southernblot analysis indicates that many of these mice contain one or fewercopies of the transgene per cell. The detection of human IgM in theserum of pIGM1 transgenics, and human IgM and IgG1 in pHC1 transgenics,provides evidence that the transgene sequences function correctly indirecting VDJ joining, transcription, and isotype switching. One of theanimals (#18) was negative for the transgene by Southern blot analysis,and showed no detectable levels of human IgM or IgG1. The second animal(#38) contained approximately 5 copies of the transgene, as assayed bySouthern blotting, and showed detectable levels of both human IgM andIgG1. The results of ELISA assays for 11 animals that developed fromtransgene injected embryos is summarized in the table below (Table 3).

                  TABLE 3                                                         ______________________________________                                        Detection of human IgM and IgG1 in the serum of transgenic                    animals by ELISA assay                                                                        approximate                                                          injected transgene                                                     animal #                                                                             transgene                                                                              copies per cell                                                                           human IgM                                                                             human IgG1                                ______________________________________                                        6      pIGM1    1           ++      -                                         7      pIGM1    0           -       -                                         9      pIGM1    0           -       -                                         10     pIGM1    0           -       -                                         12     pIGM1    0           -       -                                         15     pIGM1    10          ++      -                                         18     pHC1     0           -       -                                         19     pHC1     1           -       -                                         21     pHC1     <1          -       -                                         26     pHC1     2           ++      +                                         38     pHC1     5           ++      +                                         ______________________________________                                    

Table 3 shows a correlation between the presence of integrated transgeneDNA and the presence of transgene encoded immunoglobulins in the serum.Two of the animals that were found to contain the pHC1 transgene did notexpress detectable levels of human immunoglobulins. These were both lowcopy animals and may not have contained complete copies of thetransgenes, or the animals may have been genetic mosaics (indicated bythe <1 copy per cell estimated for animal #21), and the transgenecontaining cells may not have populated the hematopoietic lineage.Alternatively, the transgenes may have integrated into genomic locationsthat are not conducive to their expression. The detection of human IgMin the serum of pIGM1 transgenics, and human IgM and IgG1 in pHC1transgenics, indicates that the transgene sequences function correctlyin directing VDJ joining, transcription, and isotype switching.

F. cDNA clones

To assess the functionality of the pHC1 transgene in VDJ joining andclass switching, as well the participation of the transgene encodedhuman B-cell receptor in B-cell development and allelic exclusion, thestructure of immunoglobulin cDNA clones derived from transgenic mousespleen mRNA were examined. The overall diversity of the transgeneencoded heavy chains, focusing on D and J segment usage, N regionaddition, CDR3 length distribution, and the frequency of jointsresulting in functional mRNA molecules was examined. Transcriptsencoding IgM and IgG incorporating VH105 and VH251 were examined.

Polyadenylated RNA was isolated from an eleven week old male secondgeneration line-57 pHC1 transgenic mouse. This RNA was used tosynthesize oligo-dT primed single stranded cDNA. The resulting cDNA wasthen used as template for four individual PCR amplifications using thefollowing four synthetic oligonucleotides as primers: VH251 specificoligo-149, cta gct cga gtc caa gga gtc tgt gcc gag gtg cag ctg(g,a,t,c); VH105 specific o-150, gtt gct cga gtg aaa ggt gtc cag tgt gaggtg cag ctg (g,a,t,c); human gammal specific oligo-151, ggc gct cga gttcca cga cac cgt cac cgg ttc; and human mu specific oligo-152, cct gctcga ggc agc caa cgg cca cgc tgc tcg. Reaction 1 used primers 0-149 ando-151 to amplify VH251-gammal transcripts, reaction 2 used o-149 ando-152 to amplify VH251-mu transcripts, reaction 3 used o-150 and o-151to amplify VH105-gammal transcripts, and reaction 4 used o-150 and o-152to amplify VH105-mu transcripts. The resulting 0.5 kb PCR products wereisolated from an agarose gel; the μ transcript products were moreabundant than the Υ transcript products, consistent with thecorresponding ELISA data (FIG. 34). The PCR products were digested withXhoI and cloned into the plasmid pNN03. Double-stranded plasmid DNA wasisolated from minipreps of nine clones from each of the four PCRamplifications and dideoxy sequencing reactions were performed. Two ofthe clones turned out to be deletions containing no D or J segments.These could not have been derived from normal RNA splicing products andare likely to have originated from deletions introduced during PCRamplification. One of the DNA samples turned out to be a mixture of twoindividual clones, and three additional clones did not produce readableDNA sequence (presumably because the DNA samples were not clean enough).The DNA sequences of the VDJ joints from the remaining 30 clones arecompiled in Table 4. Each of the sequences are unique, indicating thatno single pathway of gene rearrangement, or single clone of transgeneexpressing B-cells is dominant. The fact that no two sequences are alikeis also an indication of the large diversity of immunoglobulins that canbe expressed from a compact minilocus containing only 2 V segments, 10 Dsegments, and 6 J segments. Both of the V segments, all six of the Jsegments, and 7 of the 10 D segments that are included in the transgeneare used in VDJ joints. In addition, both constant region genes (mu andgammal) are incorporated into transcripts. The VH105 primer turned outnot to be specific for VH105 in the reactions performed. Therefore manyof the clones from reactions 3 and 4 contained VH251 transcripts.Additionally, clones isolated from ligated reaction 3 PCR product turnedout to encode IgM rather than IgG; however this may reflectcontamination with PCR product from reaction 4 as the DNA was isolatedon the same gel. An analogous experiment, in which immunoglobulin heavychain sequences were amplified from adult human peripheral bloodlymphocytes (PBL), and the DNA sequence of the VDJ joints determined,was recently reported by Yamada et al. (J. Exp. Med. 173:395-407 (1991),which is incorporated herein by reference). We compared the data fromhuman PBL with our data from the pHC1 transgenic mouse.

    TABLE 4      - V n-D n J C      1 VH251 DHQ52 J3 γ1 TACTGTGCGAGA CGGCTAACTGGGGTTGAT GCTTTTGATATCTG     GGGCCAAGGGACAATGGTCACCGTCTCTTCAG CCTCCACCAAG     2 VH251 DN1 J4 γ1 TACTGTGCGAGA CACCGTATAGCAGCAGCTGG CTTTGACTACTGGGG     CCAGGGAACCCTGGTCACCGTCTCCTCAG CCTCCACCAAG     3 VH251 D? J6 γ1 TACTGTGCGAGA T ATTACTACTACTACTACGGTATGGACGTCTGGGGC     CAAGGGACCACGGTCACCGTCTCCTCAG CCTCCACCAAG     4 VH251 DXP'1 J6 γ1 TACTGTGCGAGA CATTACGATATTTTGACTGGTC CTACTACTACT     ACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG CCTCCACCAAG                                                                              5      VH251 DXP'1 J4 γ1 TACTGTGCGAGA CGGAGGTACTATGGTTCGGGGAGTTATTATAACGT      CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG CCTCCACCAAG                                                                              6      VH251 D? J3 γ1 TACTGTGCGAGA CGGGGGGTGTCTGAT GCTTTTGATATCTGGGGCCAAG     GGACAATGGTCACCGTCTCTTCAG CCTCCACCAAG     7 VH251 DHQ52 J3 μ TACTGTGCGAGA GCAACTGGC GCTTTTGATATCTGGGGCCAAGGGACAA     TGGTCACCGTCTCTTCAG GGAGTGCATCC     8 VH251 DHQ52 J6 μ TACTGTGCGAGA TCGGCTAACTGGGGATC CTACTACTACTACGGTATGG     ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG GGAGTGCATCC     9 VH251 -- J1 μ TACTGTGCGAGA  TACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCT     CCTCAG GGAGTGCGTCC     10 VH251 DLR2 J4 μ TACTGTGCGAGA CACGTAGCTAACTCT TTTGACTACTGGGGCCAGGGAA     CCCTGGTCACCGTCTCCTCAG GGAGTGCATCC                                                                              1     1 VH251 DXP'1 J4 μ TACTGTGCGAGA CAAATTACTATGGTTCGGGGAGTTCC CTTTGACTACT     GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG GGAGTGCATCC                                                                              1     2 VH251 D? J1 μ TACTGTGCGAGA C AATACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCG     TCTCCTCAG GGAGTGCATCC     13 VH251 DHQ52 J6 μ TACTGTGCGAGA CAAACTGGGG ACTACTACTACTACGGTATGGACGTC     TGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG GGAGTGCATCC     14 VH251 DXP'1 J6 μ TACTGTGCGAGA CATTACTATGGTTCGGGGAGTTATG ACTACTACTAC     TACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG GGAGTGCATCC     15 VH251 DXP'1 J4 γ1 TACTGTGCGAGA CAGGGAG TGGGGCCAGGGAACCCTGGTCACCG     TCTCCTCAG CCTCCACCAAG     16 VH105 DXP'1 J5 μ TACTGTGTGAGA TTCTGCGAG ACTGGTTCGACCCCTGGGGCCAGGGAA     CCCTGGTCACCGTCTCCTCAG GGAGTGCATCC                                                                              1     7 VH251 DXP'1 J4 γ1 TACTGTGCGAGA CGGAGGTACTATGGTTCGGGGAGTTATTATAACG     T CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG CCTCCACCAAG                                                                              1     8 VH251 DHQ52 J4 γ1 TACTGTGCGAGA CAAACCTGGGGAGGA GACTACTGGGGCCAGGGA     ACCCTGGTCACCGTCTCCTCAG CCTCCACCAAG     19 VH251 DK1 J6 γ1 TACTGTGCGAGA GGATATAGTGGCTACGATA ACTACTACTACGGTA     TGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG CCTCCACCAAG     20 VH251 DHQ52 J4 μ TACTGTGCGAGA CAAACTGGGGAGG ACTACTTTGACTACTGGGGCCAG     GGAACCCTGGTCACCGTCTCCTCAG GGAGTGCATCC     21 VH251 DK1 J2 γ1 TACTGTGCGAGA TATAGTGGCTACGATTAC CTACTGGTACTTCGAT     CTCTGGGGCCGTGGCACCCTGGTCACTGTCTCCTCAG CCTCCACCGAG     22 VH251 DIR2 J6 γ1 TACTGTGCGAGA GCATCCCTCCCCTCCTTTG ACTACTACGGTATG     GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG CCTCCACCAAG     23 VH251 DIR2 J4 μ TACTGTGCGAGA CGGGGTGGGGG TTTGACTACTGGGGCCAGGGAACCCT     GGTCACCGTCTCCTCAG GGAGTGCATCC     24 VH105 D? J6 μ TACTGTGTG CCGGTCGAAACT TTACTACTACTACTACGGTATGGACGTCTG     GGGCCAAGGGACCACGGTCACCGTCTCCTCAG GGAGTGCATCC     25 VH105 DXP1 J4 μ TACTGTGTGAGA GATATTTTGACTGGTTAACG TGACTACTGGGGCCAGG     GAACCCTGGTCACCGTCTCCTCAG GGAGTGCATCC     26 VH251 DN1 J3 μ TACTGTGCGAGA CATGGTATAGCAGCAGCTGGTAC TGCTTTTGATATCTG     GGGCCAAGGGACAATGGTCACCGTCTCTTCAG GGAGTGCATCC     27 VH105 DHQ52 J3 μ TACTGTGTGAGA TCAACTGGGGTTG ATGCTTTTGATATCTGGGGCCAA     GGGACAATGGTCACCGTCTCTTCAG GGAGTGCATCC     28 VH251 DN1 J4 μ TACTGTGCG GAAATAGCAGCAGCTGCC CTACTTTGACTACTGGGGCCAGG     GAACCCTGGTCACCGTCTCCTCAG GGAGTGCATCC                                                                              2     9 VH105 DN1 J4 μ TACTGTGTG TGTATAGCAGCAGCTGGTAAAGGAAACGG CTACTGGGGCCAG     GGAACCCTGGTCACCGTCTCCTCAG GGAGTGCATCC     30 VH251 DHQ52 J4 μ TACTGTGCGAGA CAAAACTGGGG TGACTACTGGGGCCAGGGAACCCTG     GTCACCGTCTCCTCAG GGAGTGCATCC

G. J seqment choice

Table 5 compared the distribution of J segments incorporated into pHC1transgene encoded transcripts to J segments found in adult human PBLimmunoglobulin transcripts. The distribution profiles are very similar,J4 is the dominant segment in both systems, followed by J6. J2 is theleast common segment in human PBL and the transgenic animal.

                  TABLE 5                                                         ______________________________________                                        J. Segment Choice                                                                        Percent usage (±3%)                                             J. Segment   HC1 transgenic                                                                            Human PBL                                            ______________________________________                                        J1           7           1                                                    J2           3           <1                                                   J3           17          9                                                    J4           44          53                                                   J5           3           15                                                   J6           26          22                                                                100%        100%                                                 ______________________________________                                    

H. D segment choice

49% (40 of 82) of the clones analyzed by Yamada et al. incorporated Dsegments that are included in the pHC1 transgene. An additional 11clones contained sequences that were not assigned by the authors to anyof the known D segments. Two of these 11 unassigned clones appear to bederived from an inversion of the DIR2 segments which is included in thepHC1 construct. This mechanism, which was predicted by Ichihara et al.(EMBO J. 7:4141 (1988)) and observed by Sanz (J. Immunol. 147:1720-1729(1991)), was not considered by Yamada et al. (J. Exp. Med. 173:395-407(1991)). Table 5 is a comparison of the D segment distribution for thepHC1 transgenic mouse and that observed for human PBL transcripts byYamada et al. The data of Yamada et al. was recompiled to include DIR2use, and to exclude D segments that are not in the pHC1 transgene. Table6 demonstrates that the distribution of D segment incorporation is verysimilar in the transgenic mouse and in human PBL. The two dominant humanD segments, DXP'1 and DN1, are also found with high frequency in thetransgenic mouse. The most dramatic dissimilarity between the twodistributions is the high frequency of DHQ52 in the transgenic mouse ascompared to the human. The high frequency of DHQ52 is reminiscent of theD segment distribution in the human fetal liver. Sanz has observed that14% of the heavy chain transcripts contained DHQ52 sequences. If Dsegments not found in pHC1 are excluded from the analysis, 31% of thefetal transcripts analyzed by Sanz contain DHQ52. This is comparable tothe 27% that we observe in the pHC1 transgenic mouse.

                  TABLE 6                                                         ______________________________________                                        D. Segment Choice                                                                        Percent usage (±3%)                                             D. Segment   HC1 transgenic                                                                            Human PBL                                            ______________________________________                                        DLR1         <1          <1                                                   DXP1         3           6                                                    DXP'1        25          19                                                   DA1          <1          12                                                   DK1          7           12                                                   DN1          12          22                                                   DIR2         7           4                                                    DM2          <1          2                                                    DLR2         3           4                                                    DHQ52        26          2                                                    ?            17          17                                                                100%        100%                                                 ______________________________________                                    

I. Functionality of VDJ joints

Table 7 shows the predicted amino acid sequences of the VDJ regions from30 clones that were analyzed from the pHC1 transgenic. The translatedsequences indicate that 23 of the 30 VDJ joints (77%) are in-frame withrespect to the variable and J segments.

                                      TABLE 7                                     __________________________________________________________________________    Functionality of V-D-J Joints                                                                FR3 CDR3                   FR4                                 __________________________________________________________________________    1 VH251                                                                             DHQ52                                                                              J3                                                                              γ1                                                                        YCAR                                                                              RLTGVDAFDI             WGQGTMVTVSSASTK                     2 VH251                                                                             DN1  J4                                                                              γ1                                                                        YCAR                                                                              HRIAAAGFDY             WGQGTLVTVSSASTK                     3 VH251                                                                             D?   J6                                                                              γ1                                                                        YCAR                                                                              YYYYYYGMDV             WGQGTTVTVSSASTK                     4 VH251                                                                             DXP'1                                                                              J6                                                                              γ1                                                                        YCAR                                                                              HYDILTGPTTTTVWTSGAKGPRSPSPQPPP                             5 VH251                                                                             DXP'1                                                                              J4                                                                              γ1                                                                        YCAR                                                                              RRYYGSGSYYNVFDY        WGQGTMVTVSSASTK                     6 VH251                                                                             D?   J3                                                                              γ1                                                                        YCAR                                                                              RGVSDAFDI              WGQGTMVTVSSASTK                     7 VH251                                                                             DHQ52                                                                              J3                                                                              μ                                                                            YCAR                                                                              ATGAFDI                WGQGTMVTVSSGSAS                     8 VH251                                                                             DHQ52                                                                              J6                                                                              μ                                                                            YCAR                                                                              SANWGSYYYYGMDV         WGQGTMVTVSSGSAS                     9 VH251                                                                             --   J1                                                                              μ                                                                            YCAR                                                                              YFQH                   WGQGTMVTVSSGSAS                     10                                                                              VH251                                                                             DLR2 J4                                                                              μ                                                                            YCAR                                                                              HVANSFDY               WGQGTMVTVSSGSAS                     11                                                                              VH251                                                                             DXP'1                                                                              J4                                                                              μ                                                                            YCAR                                                                              QITMVRGVPFDY           WGQGTMVTVSSGSAS                     12                                                                              VH251                                                                             D?   J1                                                                              μ                                                                            YCAR                                                                              QYFQH                  WGQGTMVTVSSGSAS                     13                                                                              VH251                                                                             DHQ52                                                                              J6                                                                              μ                                                                            YCAR                                                                              QTGDYYYYGMDV           WGQGTMVTVSSGSAS                     14                                                                              VH251                                                                             DXP'1                                                                              J6                                                                              μ                                                                            YCAR                                                                              HYYGSGSYDYYYYGMDV      WGQGTMVTVSSGSAS                     15                                                                              VH251                                                                             DXP'1                                                                              J4                                                                              γ1                                                                        YCVR                                                                              QGVGPGNPGHRLLSLHQ                                          16                                                                              VH105                                                                             DXP'1                                                                              J5                                                                              μ                                                                            YCAR                                                                              FWETGSTPGAREPWSPSPQGVH                                     17                                                                              VH251                                                                             DXP'1                                                                              J4                                                                              γ1                                                                        YCAR                                                                              RRYYGSGSYYNVFDY        WGQGTLVTVSSASTK                     18                                                                              VH251                                                                             DHQ52                                                                              J4                                                                              γ1                                                                        YCAR                                                                              QTWGGDY                WGQGTLVTVSSASTK                     19                                                                              VH251                                                                             DK1  J6                                                                              γ1                                                                        YCAR                                                                              GYSGYDNYYYGIHV         WGQGTTVTVSSASTK                     20                                                                              VH251                                                                             DHQ52                                                                              J4                                                                              μ                                                                            YCAR                                                                              QTGEDYFDY              WGQGTLVTVSSGSAS                     21                                                                              VH251                                                                             DK1  J2                                                                              γ1                                                                        YCAR                                                                              YSGYDYLLVLRSLGPWHPGHCLLSLHR                                22                                                                              VH251                                                                             DIR2 J6                                                                              γ1                                                                        YCAR                                                                              ASLPSFDYYGMDV          WGQGTTVTVSSASTK                     23                                                                              VH251                                                                             DIR2 J4                                                                              μ                                                                            YCAR                                                                              RGGGLTTGAREPWSPSPQGVH                                      24                                                                              VH105                                                                             D?   J6                                                                              μ                                                                            YCVP                                                                              VETLLLLLRYGRLGPRDHGHRLLRECI                                25                                                                              VH105                                                                             DXP1 J4                                                                              μ                                                                            YCVR                                                                              DILTGZRDY              WGQGTLVTVSSGSAS                     26                                                                              VH251                                                                             DM1  J3                                                                              μ                                                                            YCAR                                                                              HGIAAAGTAFDI           WGQGTMVTVSSGSAS                     27                                                                              VH105                                                                             DHQ52                                                                              J3                                                                              μ                                                                            YCVR                                                                              STGCDAFDI              WGQGTMVTVSSGSAS                     28                                                                              VH251                                                                             DN1  J4                                                                              μ                                                                            YCAE                                                                              IAAAALLZLLGPGNPGHRLLRECI                                   29                                                                              VH105                                                                             DN1  J4                                                                              μ                                                                            YCVC                                                                              IAAAGKGNGY             WGQGTLVTVSSGSAS                     30                                                                              VH251                                                                             DHQ52                                                                              J4                                                                              μ                                                                            YCAR                                                                              QNWGDY                 WGQGTLVTVSSGSAS                     __________________________________________________________________________

J. CDR3 length distribution

Table 8 compared the length of the CDR3 peptides from transcripts within-frame VDJ joints in the pHC1 transgenic mouse to those in human PBL.Again the human PBL data comes from Yamada et al. The profiles aresimilar with the transgenic profile skewed slightly toward smaller CDR3peptides than observed from human PBL. The average length of CDR3 in thetransgenic mouse is 10.3 amino acids. This is substantially the same asthe average size reported for authentic human CDR3 peptides by Sanz (J.Immunol. 147:1720-1729 (1991)).

                  TABLE 8                                                         ______________________________________                                        CDR3 Length Distribution                                                                    Percent Occurrence (±3%)                                     #amino acids in CDR3                                                                          HC1 transgenic                                                                            Human PBL                                         ______________________________________                                        3-8             26          14                                                 9-12           48          41                                                13-18           26          37                                                19-23           <1          7                                                 >23             <1          1                                                                 100%        100%                                              ______________________________________                                    

EXAMPLE 13 Rearranged Heavy Chain Transgenes

A. Isolation of Rearranged Human Heavy Chain VDJ segments.

Two human leukocyte genomic DNA libraries cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) arescreened with a 1 kb PacI/HindIII fragment of λ1.3 containing the humanheavy chain J-μ intronic enhancer. Positive clones are tested forhybridization with a mixture of the following V_(H) specificoligonucleotides: ##STR43##

Clones that hybridized with both V and J-μ probes are isolated and theDNA sequence of the rearranged VDJ segment determined.

B. Construction of rearranged human heavy chain transgenes

Fragments containing functional VJ segments (open reading frame andsplice signals) are subcloned into the plasmid vector pSP72 such thatthe plasmid derived XhoI site is adjacent to the 5' end of the insertsequence. A subclone containing a functional VDJ segment is digestedwith XhoI and PacI (PacI, a rare-cutting enzyme, recognizes a site nearthe J-m intronic enhancer), and the insert cloned into XhoI/PacIdigested pHC2 to generate a transgene construct with a functional VDJsegment, the J-μ intronic enhancer, the μ switch element, the μ constantregion coding exons, and the γ1 constant region, including the steriletranscript associated sequences, the γ1 switch, and the coding exons.This transgene construct is excised with NotI and microinjected into thepronuclei of mouse embryos to generate transgenic animals as describedabove.

EXAMPLE 14 Light Chain Transgenes

A. Construction of Plasmid vectors

1. Plasmid vector pGP1c

Plasmid vector pGP1a is digested with NotI and the followingoligonucleotides ligated in: ##STR44## The resulting plasmid, pGP1c,contains a polylinker with XmaI, XhoI, SalI, HindIII, and BamHIrestriction sites flanked by NotI sites.

2. Plasmid vector pGP1d

Plasmid vector pGP1a is digested with NotI and the followingoligonucleotides ligated in: ##STR45## The resulting plasmid, pGP1d,contains a polylinker with SalI, HindIII, ClaI, BamHI, and XhoIrestriction sites flanked by NotI sites.

B. Isolation of Jκ and Cκ clones

A human placental genomic DNA library cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) wasscreened with the human kappa light chain J region specificoligonucleotide: ##STR46## and the phage clones 136.2 and 136.5isolated. A 7.4 kb XhoI fragment that includes the Jκ1 segment wasisolated from 136.2 and subcloned into the plasmid pNNO3 to generate theplasmid clone p36.2. A neighboring 13 kb XhoI fragment that includes Jksegments 2 through 5 together with the Cκ gene segment was isolated fromphage clone 136.5 and subcloned into the plasmid pNNO3 to generate theplasmid clone p36.5. Together these two clones span the region beginning7.2 kb upstream of Jκ1 and ending 9 kb downstream of Cκ.

C. Construction of rearranged light chain transgenes

1. pCK1, a Cκ vector for expressing rearranged variable segments

The 13 kb XhoI insert of plasmid clone p36.5 containing the Cκ gene,together with 9 kb of downstream sequences, is cloned into the Sail siteof plasmid vector pGP1c with the 5' end of the insert adjacent to theplasmid XhoI site. The resulting clone, pCK1 can accept cloned fragmentscontaining rearranged VJκ segments into the unique 5' XhoI site. Thetransgene can then be excised with NotI and purified from vectorsequences by gel electrophoresis. The resulting transgene construct willcontain the human J-Cκ intronic enhancer and may contain the human 3' κenhancer.

2. pCK2, a Cκ vector with heavy chain enhancers for expressingrearranged variable segments

A 0.9 kb XbaI fragment of mouse genomic DNA containing the mouse heavychain J-μ intronic enhancer (J. Banerji et al., Cell 33:729-740 (1983))was subcloned into pUC18 to generate the plasmid pJH22.1. This plasmidwas linearized with SphI and the ends filled in with Klenow enzyme. TheKlenow treated DNA was then digested with HindIII and a 1.4 kbMluI/HindIII fragment of phage clone λ1.3 (previous example), containingthe human heavy chain J-μ intronic enhancer (Hayday et al., Nature307:334-340 (1984)), to it. The resulting plasmid, pMHE1, consists ofthe mouse and human heavy chain J-μ intronic enhancers ligated togetherinto pUC18 such that they are excised on a single BamHI/HindIIIfragment. This 2.3 kb fragment is isolated and cloned into pGP1c togenerate pMHE2. pMHE2 is digested with SalI and the 13 kb XhoI insert ofp36.5 cloned in. The resulting plasmid, pCK2, is identical to pCK1,except that the mouse and human heavy chain J-μ intronic enhancers arefused to the 3' end of the transgene insert. To modulate expression ofthe final transgene, analogous constructs can be generated withdifferent enhancers, i.e. the mouse or rat 3' kappa or heavy chainenhancer (Meyer and Neuberger, EMBO J., 8:1959-1964 (1989); Petterson etal., Nature, 344:165-168 (1990)).

3. Isolation of rearranged kappa light chain variable segments

Two human leukocyte genomic DNA libraries cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) werescreened with the human kappa light chain J region containing 3.5 kbXhoI/SmaI fragment of p36.5. Positive clones were tested forhybridization with the following Vκ specific oligonucleotide: ##STR47##Clones that hybridized with both V and J probes are isolated and the DNAsequence of the rearranged VJκ segment determined.

4. Generation of transgenic mice containing rearranged human light chainconstructs.

Fragments containing functional VJ segments (open reading frame andsplice signals) are subcloned into the unique XhoI sites of vectors pCK1and pCK2 to generate rearranged kappa light chain transgenes. Thetransgene constructs are isolated from vector sequences by digestionwith NotI. Agarose gel purified insert is microinjected into mouseembryo pronuclei to generate transgenic animals. Animals expressinghuman kappa chain are bred with heavy chain minilocus containingtransgenic animals to generate mice expressing fully human antibodies.

Because not all VJκ combinations may be capable of forming stableheavy-light chain complexes with a broad spectrum of different heavychain VDJ combinations, several different light chain transgeneconstructs are generated, each using a different rearranged VJk clone,and transgenic mice that result from these constructs are bred withheavy chain minilocus transgene expressing mice. Peripheral blood,spleen, and lymph node lymphocytes are isolated from double transgenic(both heavy and light chain constructs) animals, stained withfluorescent antibodies specific for human and mouse heavy and lightchain immunoglobulins (Pharmingen, San Diego, Calif.) and analyzed byflow cytometry using a FACScan analyzer (Becton Dickinson, San Jose,Calif.). Rearranged light chain transgenes constructs that result in thehighest level of human heavy/light chain complexes on the surface of thehighest number of B cells, and do not adversely affect the immune cellcompartment (as assayed by flow cytometric analysis with B and T cellsubset specific antibodies), are selected for the generation of humanmonoclonal antibodies.

D. Construction of unrearranged light chain minilocus transgenes

1. pJCK1, a Jκ, Cκ containing vector for constructing minilocustransgenes

The 13 kb Cκ containing XhoI insert of p36.5 is treated with Klenowenzyme and cloned into HindIII digested, Klenow-treated, plasmid pGP1d.A plasmid clone is selected such that the 5' end of the insert isadjacent to the vector derived ClaI site. The resulting plasmid,p36.5-1d, is digested with ClaI and Klenow-treated. The Jκ1 containing7.4 kb XhoI insert of p36.2 is then Klenow-treated and cloned into theClaI, Klenow-treated p36.5-1d. A clone is selected in which the p36.2insert is in the same orientation as the p36.5 insert. This clone, pJCK1(FIG. 34), contains the entire human Jκ region and Cκ, together with 7.2kb of upstream sequences and 9 kb of downstream sequences. The insertalso contains the human J-Cκ intronic enhancer and may contain a human3'κ enhancer. The insert is flanked by a unique 3' SalI site for thepurpose of cloning additional 3' flanking sequences such as heavy chainor light chain enhancers. A unique XhoI site is located at the 5' end ofthe insert for the purpose of cloning in unrearranged Vκ gene segments.The unique SalI and XhoI sites are in turn flanked by NotI sites thatare used to isolate the completed transgene construct away from vectorsequences.

2. Isolation of unrearranged Vκ gene segments and generation oftransgenic animals expressing human Ig light chain protein

The Vκ specific oligonucleotide, oligo-65 (discussed above), is used toprobe a human placental genomic DNA library cloned into the phage vector1EMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.).Variable gene segments from the resulting clones are sequenced, andclones that appear functional are selected. Criteria for judgingfunctionality include: open reading frames, intact splice acceptor anddonor sequences, and intact recombination sequence. DNA fragmentscontaining selected variable gene segments are cloned into the uniqueXhoI site of plasmid pJCK1 to generate minilocus constructs. Theresulting clones are digested with NotI and the inserts isolated andinjected into mouse embryo pronuclei to generate transgenic animals. Thetransgenes of these animals will undergo V to J joining in developingB-cells. Animals expressing human kappa chain are bred with heavy chainminilocus containing transgenic animals to generate mice expressingfully human antibodies.

EXAMPLE 15 Genomic Heavy Chain Human Ig Transgene

This Example describes the cloning of a human genomic heavy chainimmunoglobulin transgene which is then introduced into the murinegermline via microinjection into zygotes or integration in ES cells.

Nuclei are isolated from fresh human placental tissue as described byMarzluff, W. F., et al. (1985), Transcription and Translation: APractical Approach, B. D. Hammes and S. J. Higgins, eds., pp. 89-129,IRL Press, Oxford). The isolated nuclei (or PBS washed humanspermatocytes) are embedded in 0.5% low melting point agarose blocks andlysed with 1 mg/ml proteinase K in 500 mM EDTA, 1% SDS for nuclei, orwith 1 mg/ml proteinase K in 500 mM EDTA, 1% SDS, 10 mM DTT forspermatocytes at 50° C. for 18 hours. The proteinase K is inactivated byincubating the blocks in 40 μg/ml PMSF in TE for 30 minutes at 50° C.,and then washing extensively with TE. The DNA is then digested in theagarose with the restriction enzyme NotI as described by M. Finney inCurrent Protocols in Molecular Bioloqy (F. Ausubel et al., eds. JohnWiley & Sons, Supp. 4, 1988, e.g., Section 2.5.1).

The NotI digested DNA is then fractionated by pulsed field gelelectrophoresis as described by Anand et al., Nuc. Acids Res.17:3425-3433 (1989). Fractions enriched for the NotI fragment areassayed by Southern hybridization to detect one or more of the sequencesencoded by this fragment. Such sequences include the heavy chain Dsegments, J segments, and γ1 constant regions together withrepresentatives of all 6 V_(H) families (although this fragment isidentified as 670 kb fragment from HeLa cells by Berman et al. (1988),supra., we have found it to be an 830 kb fragment from human placentaland sperm DNA). Those fractions containing this NotI fragment areligated into the NotI cloning site of the vector pYACNN as described(McCormick et al., Technique 2:65-71 (1990)). Plasmid pYACNN is preparedby digestion of pYACneo (Clontech) with EcoRI and ligation in thepresence of the oligonucleotide 5'--AAT TGC GGC CGC--3'.

YAC clones containing the heavy chain NotI fragment are isolated asdescribed by Traver et al., Proc. Natl. Acad. Sci. USA, 86:5898-5902(1989). The cloned NotI insert is isolated from high molecular weightyeast DNA by pulse field gel electrophoresis as described by M. Finney,op. cit. The DNA is condensed by the addition of 1 mM spermine andmicroinjected directly into the nucleus of single cell embryospreviously described. Alternatively, the DNA is isolated by pulsed fieldgel electrophoresis and introduced into ES cells by lipofection (Gnirkeet al., EMBO J. 10:1629-1634 (1991)), or the YAC is introduced into EScells by spheroplast fusion.

EXAMPLE 16 Discontinuous Genomic Heavy Chain Ig Transgene

An 85 kb SpeI fragment of human genomic DNA, containing V_(H) 6, Dsegments, J segments, the μ constant region and part of the γ constantregion, has been isolated by YAC cloning essentially as described inExample 1. A YAC carrying a fragment from the germline variable region,such as a 570 kb NotI fragment upstream of the 670-830 kb NotI fragmentdescribed above containing multiple copies of V₁ through V₅, is isolatedas described. (Berman et al. (1988), supra. detected two p570 kb NotIfragments, each containing multiple V segments.) The two fragments arecoinjected into the nucleus of a mouse single cell embryo as describedin Example 1.

Typically, coinjection of two different DNA fragments result in theintegration of both fragments at the same insertion site within thechromosome. Therefore, approximately 50% of the resulting transgenicanimals that contain at least one copy of each of the two fragments willhave the V segment fragment inserted upstream of the constant regioncontaining fragment. Of these animals, about 50% will carry out V to DJjoining by DNA inversion and about 50% by deletion, depending on theorientation of the 570 kb NotI fragment relative to the position of the85 kb SpeI fragment. DNA is isolated from resultant transgenic animalsand those animals found to be containing both transgenes by Southernblot hybridization (specifically, those animals containing both multiplehuman V segments and human constant region genes) are tested for theirability to express human immunoglobulin molecules in accordance withstandard techniques.

EXAMPLE 17 Identification of functionally rearranged variable regionsequences in transgenic B cells

An antigen of interest is used to immunize (see Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988)) amouse with the following genetic traits: homozygosity at the endogenoushaving chain locus for a deletion of J_(H) (Examples 10); hemizygous fora single copy of unrearranged human heavy chain minilocus transgene(examples 5 and 14); and hemizygous for a single copy of a rearrangedhuman kappa light chain transgene (Examples 6 and 14).

Following the schedule of immunization, the spleen is removed, andspleen cells used to generate hybridomas. Cells from an individualhybridoma clone that secretes antibodies reactive with the antigen ofinterest are used to prepare genomic DNA. A sample of the genomic DNA isdigested with several different restriction enzymes that recognizeunique six base pair sequences, and fractionated on an agarose gel.Southern blot hybridization is used to identify two DNA fragments in the2-10 kb range, one of which contains the single copy of the rearrangedhuman heavy chain VDJ sequences and one of which contains the singlecopy of the rearranged human light chain VJ sequence. These twofragments are size fractionated on agarose gel and cloned directly intopUC18. The cloned inserts are then subcloned respectively into heavy andlight chain expression cassettes that contain constant region sequences.

The plasmid clone pγel (Example 12) is used as a heavy chain expressioncassette and rearranged VDJ sequences are cloned into the XhoI site. Theplasmid clone pCK1 is used as a light chain expression cassette andrearranged VJ sequences are cloned into the XhoI site. The resultingclones are used together to transfect SP₀ cells to produce antibodiesthat react with the antigen of interest (Co. et al., Proc. Natl. Acad.Sci. USA 88:2869 (1991), which is incorporated herein by reference).

Alternatively, mRNA is isolated from the cloned hybridoma cellsdescribed above, and used to synthesize cDNA. The expressed human heavyand light chain VDJ and VJ sequence are then amplified by PCR and cloned(Larrick et al., Biol. Technology, 7:934-938 (1989)). After thenucleotide sequence of these clones has been determined,oligonucleotides are synthesized that encode the same polypeptides, andsynthetic expression vectors generated as described by Queen et al.,Proc. Natl. Acad. Sci. USA., 84:5454-5458 (1989).

Immunization of Transgenic Animals with Complex Antigens

The following experiment demonstrates that transgenic animals can besuccessfully immunized with complex antigens such as those on human redblood cells and respond with kinetics that are similar to the responsekinetics observed in normal mice.

Blood cells generally are suitable immunogens and comprise manydifferent types of antigens on the surface of red and white blood cells.

Immunization with human blood

Tubes of human blood from a single donor were collected and used toimmunize transgenic mice having functionally disrupted endogenous heavychain loci (JH^(D)) and harboring a human heavy chain minigene construct(HC1); these mice are designated as line 112. Blood was washed andresuspended in 50 mls Hanks' and diluted to 1×108 cells/ml 0.2 mls(2×10⁷ cells) were then injected interperitoneally using a 28 gaugeneedle and 1 cc syringe. This immunization protocol was repeatedapproximately weekly for 6 weeks. Serum titers were monitored by takingblood from retro-orbital bleeds and collecting serum and later testingfor specific antibody. A pre-immune bleed was also taken as a control.On the very last immunization, three days before these animals weresacrificed for serum and for hybridomas, a single immunization of 1×10⁷cells was given intravenously through the tail to enhance the productionof hybridomas.

                  TABLE 9                                                         ______________________________________                                        Animals                                                                       Mouse ID      Line   Sex      HC1-112                                                                              JHD                                      ______________________________________                                        1     2343        112    M      +      ++                                     2     2344        112    M      -      +                                      3     2345        112    F      -      +                                      4     2346        112    F      -      ++                                     5     2347        112    F      -      ++                                     6     2348        112    F      +      ++                                     7     2349        112    F      -      +                                      ______________________________________                                         Mice #2343 and 2348 have a desired phenotype: human heavy chain mini-gene     transgenic on heavy chain knock-out background.

Generation of Hybridomas

Hybridomas were generated by fusing mouse spleen cells of approximately16 week-old transgenic mice (Table 9) that had been immunized asdescribed (supra) to a fusion partner consisting of the non-secretingHAT-sensitive myeloma cell line, X63 Ag8.653. Hybridoma clones werecultivated and hybridoma supernatants containing immunoglobulins havingspecific binding affinity for blood cell antigens were identified, forexample, by flow cytometry.

Flow cytometry

Serum and hybridoma supernatants were tested using flow cytometry. Redblood cells from the donor were washed 4X in Hanks' balanced saltsolution and 50,000 cells were placed in 1.1 ml polypropylenemicrotubes. Cells were incubated with antisera or supernatant from thehybridomas for 30 minutes on ice in staining media (1x RPMI 1640 mediawithout phenol red or biotin (Irvine Scientific) 3% newborn calf serum,0.1% Na azide). Controls consisted of littermate mice with othergenotypes. Cells were then washed by centrifugation at 4° C. in SorvallRT600B for 5-10 minutes at 1000 rpm. Cells were washed two times andthen antibody detected on the cell surface with a fluorescent developingreagent. Two monoclonal reagents were used to test. One was aFITC-labeled mouse anti-human μ heavy chain antibody (Pharmagen, SanDiego, Calif.) and the other was a PE-labeled rat anti-mouse kappa lightchain (Becton-Dickenson, San Jose, Calif.). Both of these reagents gavesimilar results. Whole blood (red blood cells and white blood cells) andwhite blood cells alone were used as target cells. Both sets gavepositive results.

Serum of transgenic mice and littermate controls was incubated witheither red blood cells from the donor, or white blood cells from anotherindividual, washed and then developed with anti-human IgM FITC labeledantibody and analyzed in a flow cytometer. Results showed that serumfrom mice that are transgenic for the human mini-gene locus (mice 2343and 2348) show human IgM reactivity whereas all littermate animals(2344, 2345, 2346, 2347) do not. Normal mouse serum (NS) and phosphatebuffer saline (PBS) were used as negative controls. Red blood cells wereungated and white blood cells were gated to include only lymphocytes.Lines are drawn on the x and y axis to provide a reference. Flowcytometry was performed on 100 supernatants from fusion 2348. Foursupernatants showed positive reactivity for blood cell antigens.

EXAMPLE 18 Reduction of Endogenous Mouse Immunoglobulin Expression byAntisense RNA

A. Vector for Expression of Antisense Ig Sequences

1. Construction of the cloning vector pGP1h

The vector pGP1b (referred to in a previous example) is digested withXhoI and BamHI and ligated with the following oligonucleotides:##STR48## to generate the plasmid pGP1h. This plasmid contains apolylinker that includes the following restriction sites: NotI, EcoRI,BglII, Asp718, XhoI, BamHI, HindIII, NotI.

Construction of pBCE1.

A 0.8 kb XbaI/BglII fragment of pVH251 (referred to in a previousexample), that includes the promoter leader sequence exon, first intron,and part of the second exon of the human VH-V family immunoglobulinvariable gene segment, was inserted into XbaI/BglII digested vectorpNN03 to generate the plasmid pVH251.

The 2.2 kb BamHI/EcoRI DNA fragment that includes the coding exons ofthe human growth hormone gene (hGH; Seeburg, (1982) DNA 1:239-249) iscloned into BglII/EcoRI digested pGH1h. The resulting plasmid isdigested with BamHI and the BamHI/BglII of pVH251N is inserted in thesame orientation as the hGH gene to generate the plasmid pVhgh.

A 0.9 kb XbaI fragment of mouse genomic DNA containing the mouse heavychain J-μ intronic enhancer (Banerji et al., (1983) Cell 33:729-740) wassubcloned into pUC18 to generate the plasmid pJH22.1. This plasmid waslinearized with SphI and the ends filled in with klenow enzyme. Theklenow treated DNA was then digested with HindIII and a 1.4 kbMluI(klenow)/HindIII fragment of phage clone λ1.3 (previous example),containing the human heavy chain J-μ intronic enhancer (Hayday et al.,(1984) Nature 307:334-340), to it. The resulting plasmid, pMHE1,consists of the mouse and human heavy chain J-μ intron enhancers ligatedtogether into pUC18 such that they can be excised on a singleBamHI/HindIII fragment.

The BamHI/HindIII fragment of pMHE1 is cloned into BamHI/HindIII cutpVhgh to generate the B-cell expression vector pBCE1. This vector,depicted in FIG. 36, contains unique XhoI and Asp718 cloning sites intowhich antisense DNA fragments can be cloned. The expression of theseantisense sequences is driven by the upstream heavy chainpromoter-enhancer combination the downstream hGH gene sequences providepolyadenylation sequences in addition to intron sequences that promotethe expression of transgene constructs. Antisense transgene constructsgenerated from pBCE1 can be separated from vector sequences by digestionwith NotI.

B. An IgM antisense transgene construct.

The following two oligonucleotides: ##STR49## are used as primers forthe amplification of mouse IgM constant region sequences by polymerasechain reaction (PCR) using mouse spleen cDNA as a substrate. Theresulting 0.3 kb PCR product is digested with Asp718 and XhoI and clonedinto Asp718/XhoI digested pBCE1 to generate the antisense transgeneconstruct pMAS1. The purified NotI insert of pMAS1 is microinjected intothe pronuclei of half day mouse embryos--alone or in combination withone or more other transgene constructs--to generate transgenic mice.This construct expresses an RNA transcript in B-cells that hybridizeswith mouse IgM mRNA, thus down-regulating the expression of mouse IgMprotein. Double transgenic mice containing pMAS1 and a human heavy chaintransgene minilocus such as pHC1 (generated either by coinjection ofboth constructs or by breeding of singly transgenic mice) will expressthe human transgene encoded Ig receptor on a higher percentage of B-cellthan mice transgenic for the human heavy chain minilocus alone. Theratio of human to mouse Ig receptor expressing cells is due in part tocompetition between the two populations for factors and cells thatpromoter B-cell differentiation and expansion. Because the Ig receptorplays a key role in B-cell development, mouse Ig receptor expressingB-cells that express reduced levels of IgM on their surface (due tomouse Ig specific antisense down-regulation) during B-cell developmentwill not compete as well as cells that express the human receptor.

C. An IgKappa antisense transgene construct.

The following two oligonucleotides: ##STR50## are used as primers forthe amplification of mouse IgKappa constant region sequences bypolymerase chain reaction (PCR) using mouse spleen cDNA as a substrate.The resulting 0.3 kb PCR product is digested with Asp718 and XhoI andcloned into Asp718/XhoI digested pBCE1 to generate the antisensetransgene construct pKAS1. The purified NotI insert of pKAS1 ismicroinjected into the pronuclei of half day mouse embryos--alone or incombination with one or more other transgene constructs--to generatetransgenic mice. This construct expresses an RNA transcript in B-cellsthat hybridizes with mouse IgK mRNA, thus down-regulating the expressionof mouse IgK protein as described above for pMAS1.

EXAMPLE 19

This example demonstrates the successful immunization and immuneresponse in a transgenic mouse of the present invention.

Immunization of Mice

Keyhole limpet hemocyanin conjugated with greater than 400 dinitrophenylgroups per molecule (Calbiochem, La. Jolla, Calif.) (KLH-DNP) was alumprecipitated according to a previously published method (PracticalImmunology, L. Hudson and F. C. Hay, Blackwell Scientific (Pubs.), p. 9,1980). Four hundred μg of alum precipitated KLH-DNP along with 100 μgdimethyldioctadecyl Ammonium Bromide in 100 μL of phosphate bufferedsaline (PBS) was injected intraperitoneally into each mouse. Serumsamples were collected six days later by retro-orbital sinus bleeding.

Analysis of Human Antibody Reactivity in Serum

Antibody reactivity and specificity were assessed using an indirectenzyme-linked immunosorbent assay (ELISA). Several target antigens weretested to analyze antibody induction by the immunogen. Keyhole limpethemocyanin (Calbiochem) was used to identify reactivity against theprotein component, bovine serum albumin-DNP for reactivity against thehapten and/or modified amino groups, and KLH-DNP for reactivity againstthe total immunogen. Human antibody binding to antigen was detected byenzyme conjugates specific for IgM and IgG sub-classes with no crossreactivity to mouse immunoglobulin. Briefly, PVC microtiter plates werecoated with antigen drying overnight at 37° C. of 5 μg/mL protein inPBS. Serum samples diluted in PBS, 5% chicken serum, 0.5% Tween-20 wereincubated in the wells for 1 hour at room temperature, followed byanti-human IgG Fc and IgG F(ab')-horseradish peroxidase or anti-humanIgM Fc-horseradish peroxidase in the same diluent. After 1 hour at roomtemperature enzyme activity was assessed by addition of ABTS substrate(Sigma, St. Louis, Mo.) and read after 30 minutes at 415-490 nm.

Human Heavy Chain Participation in Immune Response in Transgenic Mice

FIG. 37 illustrates the response of three mouse littermates toimmunization with KLH-DNP. Mouse number 1296 carried the human IgM andIgG unrearranged transgene and was homozygous for mouse Ig heavy chainknockout. Mouse number 1299 carried the transgene on a non-knockoutbackground, while mouse 1301 inherited neither of these sets of genes.Mouse 1297, another littermate, carried the human transgene and washemizygous with respect to mouse heavy chain knockout. It was includedas a non-immunized control.

The results demonstrate that both human IgG and IgM responses weredeveloped to the hapten in the context of conjugation to protein. HumanIgM also developed to the KLH molecule, but no significant levels ofhuman IgG were present at this time point. In pre-immunization serumsamples from the same mice, titers of human antibodies to the sametarget antigens were insignificant.

EXAMPLE 20

This example demonstrates the successful immunization with a humanantigen and immune response in a transgenic mouse of the presentinvention, and provides data demonstrating that nonrandom somaticmutation occurs in the variable region sequences of the human transgene.

Demonstration of antibody responses comprising human immunoglobulinheavy chains against a human glycoprotein antigen

Transgenic mice used for the experiment were homozygous for functionallydisrupted murine immunoglobulin heavy chain loci produced byintroduction of a transgene at the joining (J) region (Supra) resultingin the absence of functional endogenous (murine) heavy chain production.The transgenic mice also harbored at least one complete unrearrangedhuman heavy chain mini-locus transgene, (HC1, supra), which included asingle functional V_(H) gene (V_(H) 251), human μ constant region gene,and human γ1 constant region gene. Transgenic mice shown to expresshuman immunoglobulin transgene products (supra) were selected forimmunization with a human antigen to demonstrate the capacity of thetransgenic mice to make an immune response against a human antigenimmunization. Three mice of the HC1-26 line and three mice of the HC1-57line (supra) were injected with human antigen.

One hundred μg of purified human carcinoembryonic antigen (CEA)insolubilized on alum was injected in complete Freund's adjuvant on Day0, followed by further weekly injections of alum-precipitated CEA inincomplete Freund's adjuvant on Days 7, 14, 21, and 28. Serum sampleswere collected by retro-orbital bleeding on each day prior to injectionof CEA. Equal volumes of serum were pooled from each of the three micein each group for analysis.

Titres of human μ chain-containing immunoglobulin and human γchain-containing immunoglobulin which bound to human CEA immobilized onmicrotitre wells were determined by ELISA assay. Results of the ELISAassays for human μ chain-containing immunoglobulins and human γchain-containing immmunoglbulins are shown in FIGS. 38 and 39,respectively. Significant human μ chain Ig titres were detected for bothlines by Day 7 and were observed to rise until about Day 21. For human γchain Ig, significant titres were delayed, being evident first for lineHC1-57 at Day 14, and later for line HC1-26 at Day 21. Titres for humanγ chain Ig continued to show an increase over time during the course ofthe experiment. The observed human μ chain Ig response, followed by aplateau, combined with a later geveloping γ chain response whichcontinues to rise is characteristic of the pattern seen with affinitymaturation. Analysis of Day 21 samples showed lack of reactivity to anunrelated antigen, keyhole limpet hemocyanin (KLC), indicating that theantibody response was directed against CEA in a specific manner.

These data indicate that animals transgenic for human unrearrangedimmunoglobulin gene loci: (1) can respond to a human antigen (e.g., thehuman glycoprotein, CEA), (2) can undergo isotype switching ("classswitching) as exemplified by the observed μ to γ class switch, and (3)exhibit characteristics of affinity maturation in their humoral immuneresponses. In general, these data indicate: (1) the human Ig transgenicmice have the ability to induce heterologous antibody production inresponse to a defined antigen, (2) the capacity of a single transgeneheavy chain variable region to respond to a defined antigen, (3)response kinetics over a time period typical of primary and secondaryresponse development, (4) class switching of a transgene-encoded humoralimmune response from IgM to IgG, and (5) the capacity of transgenicanimal to produce human-sequence antibodies against a human antigen.

Demonstration of somatic mutation in a human heavy chain transgeneminilocus.

Line HC1-57 transgenic mice, containing multiple copies of the HC1transgene, were bred with immunoglobulin heavy chain deletion mice toobtain mice that contain the HC1 transgene and contain disruptions atboth alleles of the endogenous mouse heavy chain (supra). These miceexpress human mu and gammal heavy chains together with mouse kappa andlambda light chains (supra). One of these mice was hyperimmunizedagainst human carcinoembryonic antigen by repeated intraperitonealinjections over the course of 1.5 months. This mouse was sacrificed andlymphoid cells isolated from the spleen, inguinal and mesenteric lymphnodes, and peyers patches. The cells were combined and total RNAisolated. First strand cDNA was synthesized from the RNA and used as atemplate for PCR amplification with the following 2 oligonucleotideprimers: ##STR51##

These primers specifically amplify VH251/gammal cDNA sequences. Theamplified sequences were digested with XhoI and cloned into the vectorpNN03. DNA sequence from the inserts of 23 random clones is shown inFIG. 40; sequence variations from germline sequence are indicated, dotsindicate sequence is identical to germline. Comparison of the cDNAsequences with the germline sequence of the VH251 transgene reveals that3 of the clones are completely unmutated, while the other 20 clonescontain somatic mutations. One of the 3 non-mutated sequences is derivedfrom an out-of-frame VDJ joint. Observed somatic mutations at specificpositions of occur at similar frequencies and in similar distributionpatterns to those observed in human lymphocytes (Cai et al. (1992) J.Exp. Med. 176: 1073, incorporated herein by reference). The overallfrequency of somatic mutations is approximately 1%; however, thefrequency goes up to about 5% within CDR1, indicating selection foramino acid changes that affect antigen binding. This demonstratesantigen driven affinity maturation of the human heavy chain sequences.

EXAMPLE 21

This example demonstrates the successful formation of a transgene byco-introduction of two separate polynucleotides which recombine to forma complete human light chain minilocus transgene.

Generation of an unrearranged light chain minilocus transgene byco-injection of two overlapping DNA fragments

1. Isolation of unrearranged functional V.sub.κ gene segments vk65.3,vk65.5, vk65.8 and vk65.15

The V.sub.κ specific oligonucleotide, oligo-65 (5'-agg ttc agt ggc agtggg tct ggg aca gac ttc act ctc acc atc agc-3'), was used to probe ahuman placental genomic DNA library cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.). DNAfragments containing V.sub.κ segments from positive phage clones weresubcloned into plasmid vectors. Variable gene segments from theresulting clones are sequenced, and clones that appear functional wereselected. Criteria for judging functionality include: open readingframes, intact splice acceptor and donor sequences, and intactrecombination sequence. DNA sequences of 4 functional V.sub.κ genesegments (vk65.3, vk65.5, vk65.8, and vk65.15) from 4 different plasmidclones isolated by this procedure are shown in FIGS. 41-44. The fourplasmid clones, p65.3f, p65.5gl, p65.8, and p65.15f, are describedbelow.

(1 a) p65.3f

A 3 kb Xba fragment of phage clone λ65.3 was subcloned into pUC19 sothat the vector derived SalI site was proximal to the 3' end of theinsert and the vector derived BamHI site 5'. The 3 kb BamHI/SalI insertof this clone was subcloned into pGP1f to generate p65.3f.

(1 b) p65.5g1

A 6.8 kb EcoRI fragment of phage clone λ65.5 was subcloned into pGP1f sothat the vector derived XhoI site is proximal to the 5' end of theinsert and the vector derived SalI site 3'. The resulting plasmid isdesignated p65.5g1 .

(1 c) p65.8

A 6.5 kb HindIII fragment of phage clone λ65.8 was cloned into pSP72 togenerate p65.8.

(1 d) p65.15f

A 10 kb EcoRI fragment of phage clone λ65.16 was subcloned into pUC18 togenerate the plasmid p65.15.3. The V₇₈ gene segment within the plasmidinsert was mapped to a 4.6 kb EcoRI/HindIII subfragment, which wascloned into pGP1f. The resulting clone, p65.15f, has unique XhoI andSalI sites located at the respective 5' and 3' ends of the insert.

2. pKV4

The XhoI/SalI insert of p65.8 was cloned into the XhoI site of p65.15fto generate the plasmid pKV2. The XhoI/SalI insert of p65.5g1 was clonedinto the XhoI site of pKV2 to generate pKV3. The XhoI/SalI insert ofpKV3 was cloned into the XhoI site of p65.3f to generate the plasmidpKV4. This plasmid contains a single 21 kb XhoI/SalI insert thatincludes 4 functional V.sub.κ gene segments. The entire insert can alsobe excised with NotI.

3. pKC1B

(3 a) pKcor

Two XhoI fragments derived from human genomic DNA phage λ clones weresubcloned into plasmid vectors. The first, a 13 kb J.sub.κ 2-J.sub.κ5/C.sub.κ containing fragment, was treated with Klenow enzyme and clonedinto HindIII digested, Klenow treated, plasmid pGP1d. A plasmid clone(pK-31) was selected such that the 5' end of the insert is adjacent tothe vector derived ClaI site. The second XhoI fragment, a 7.4 kb pieceof DNA containing J.sub.κ 1 was cloned into XhoI/SalI-digested pSP72,such that the 3' insert XhoI site was destroyed by ligation to thevector SalI site. The resulting clone, p36.2s, includes an insertderived ClaI site 4.5 kb upstream of J.sub.κ 1 and a polylinker derivedClaI site downstream in place of the naturally occurring XhoI sitebetween J.sub.κ 1 and J.sub.κ 2. This clone was digested with ClaI torelease a 4.7 kb fragment which was cloned into ClaI digested pK-31 inthe correct 5' to 3' orientation to generate a plasmid containing all 5human J.sub.κ segments, the human intronic enhancer human C.sub.κ, 4.5kb of 5' flanking sequence, and 9 kb of 3' flanking sequence. Thisplasmid, pKcor, includes unique flanking XhoI and SalI sites on therespective 5' and 3' sides of the insert.

(3 b) pKcorB

A 4 kb BamHI fragment containing the human 3' kappa enhancer (Judde,J.-G. and Max, E. E. (1992) Mol. Cell. Biol. 12: 5206, incorporatedherein by reference) was cloned into pGP1f such that the 5' end isproximal to the vector XhoI site. The resulting plasmid, p24Bf, was cutwith XhoI and the 17.7 kb XhoI/SalI fragment of pKcor cloned into it inthe same orientation as the enhancer fragment. The resulting plasmid,pKcorB, includes unique XhoI and SalI sites at the 5' and 3' ends of theinsert respectively.

(3 c) pKC1B

The XhoI/SalI insert of pKcorB was cloned into the SalI site of p65.3fto generate the light-chain minilocus-transgene plasmid pKC1B. Thisplasmid includes a single functional human V_(K) segment, all 5 humanJ.sub.κ segments, the human intronic enhancer, human C.sub.κ, and thehuman 3' kappa enhancer. The entire 25 kb insert can be isolated by NotIdigestion.

4. Co4

The two NotI inserts from plasmids pKV4 and pKC1B were mixed at aconcentration of 2.5 μg/ml each in microinjection buffer, andco-injected into the pronuclei of half day mouse embryos as described inprevious examples. Resulting transgenic animals contain transgeneinserts (designated Co4, product of the recombination shown in FIG. 44)in which the two fragments co-integrated. The 3' 3 kb of the pKV4 insertand the 5'3 kb of the pKC1B insert are identical. Some of theintegration events will represent homologous recombinations between thetwo fragments over the 3 kb of shared sequence. The Co4 locus willdirect the expression of a repertoire of human sequence light chains ina transgenic mouse.

The foregoing description of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in light of the above teaching.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

What is claimed is:
 1. A method for producing heterologousimmunoglobulins from a transgenic mouse, the methodcomprising:contacting said transgenic mouse with a pre-selected antigen,said transgenic mouse having a genome comprising germline copies of atleast one transgene having human sequences V_(H) segment genes, human Dsegment genes, and human J_(H) segment genes, wherein the transgeneundergoes isotype switching from a transgene-encoded mu isotype to atransgene-encoded downstream human gamma isotype in vivo, wherein thenumber of nucleotides between the gene segments within the transgenethat encode said mu and said human gamma isotypes is less than thenumber of nucleotides between the gene segments encoding human mu andsaid human gamma isotype in the human germline; and, collectingheterologous human gamma immunoglobulins which bind to said preselectedantigen.
 2. A method according to claim 1, wherein the transgenic mousehas a genome comprising germline copies of at least one human lightchain immunoglobulin transgene, wherein said light chain is a kappalight chain.
 3. A method according to claim 1, wherein the transgene isan unrearranged human heavy chain transgene comprising two human V_(H)gene segments, eight human D gene segments, six human J_(H) genesegments, a human J-mu enhancer, a human mu switch region, a completeset of human mu exons, a human sterile transcript promoter, a humangamma switch region, a complete set of human gamma C_(H) exons, and aheavy chain 3' enhancer, and wherein said unrearranged human heavy chaintransgene lacks nonhuman V_(H) gene segments, nonhuman D gene segments,and nonhuman J_(H) gene segments, and wherein B lymphocytes of saidtransgenic mouse rearrange said unrearranged human heavy chain transgeneby V-D-J joining to produce a V-D-J gene joined in-frame encoding ahuman heavy chain variable region which is alternately expressed inpolypeptide linkage by isotype switching to the mu and gamma constantregion encoded on said transgene.
 4. A method according to claim 3,wherein said V-D-J gene joined in-frame encodes a human heavychainvariable region expressed in polypeptide linkage to the human gammaconstant region encoded on said transgene.
 5. A method according toclaim 3, wherein said V-D-J gene joined in-frame comprises a human DIR2gene sequence.
 6. A method of claim 3, wherein said unrearranged humanheavy chain transgene comprises a NotI insert of pHC1.
 7. A method ofclaim 3, wherein said transgene comprises human VH gene segments VH251and VH105.
 8. A method of claim 3, wherein said transgenic mousecomprises an integrated copy of a NotI insert of pHC1, wherein saidtransgenic mouse expresses human mu or human gamma-1 chains in serum asa result of isotype switching; each human mu or human gamma-1 chaincomprising a variable region having a polypeptide sequence encoded by ahuman V_(H) gene segment, a human D segment, and a human J_(H) genesegment, said V_(H), D and J_(H) segments joined inframe.
 9. A methodaccording to claim 3, wherein said transgene further comprises a 5.3 kbHindIII fragment of a human gamma-1 heavy chain gene region, said 5.3 kbHindIII fragment having the human gamma-1 switch region and the firstexon of the pre-switch sterile transcript.
 10. A method according toclaim 9, wherein the transgene further comprises a 0.7 kb XbaI/HindIIIfragment of a human heavy chain region, said 0.7 kb XbaI/HindIIIfragment having the sequences immediately upstream of the 5.3 kb humangamma-1 switch region.
 11. A method according to claim 10, wherein thetransgene further comprises a 3.1 kb XbaI fragment having the sequencesimmediately upstream of said 0.7 kb XbaI/HindIII fragment of the humanheavy chain region.
 12. A method according to claim 3, wherein saidtransgene comprises a human gamma-1 constant region including theassociated switch region and sterile transcript associated exon,together with approximately 4 kb flanking sequences upstream of thesterile transcript initiation site, and a heavy chain 3' enhancer thatcan be PCR amplified with the following oligonucleotide primers:##STR52##
 13. A method according to claim 1, wherein said transgenecomprises a NotI insert of pHC1 and wherein said transgenic mouseexpresses both human mu and human gamma-1 chains in serum.
 14. A methodaccording to claim 1, wherein said step of collecting heterologousimmunoglobulins comprises collecting a serum containing human gamma-1chains.